Compounds and methods for treating bacterial infections

ABSTRACT

The present invention encompasses compounds and methods for treating urinary tract infections.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/570,322 filed Dec.15, 2014, which is a divisional of U.S. Ser. No. 13/453,991 filed Apr.23, 2012, now U.S. Pat. No. 8,937,167, which is a continuation-in-partof PCT application number PCT/US2010/053848 filed Oct. 22, 2010, whichclaims priority of U.S. provisional application No. 61/254,135, filedOct. 22, 2009; U.S. provisional application No. 61/384,535, filed Sep.20, 2010; and U.S. provisional application No. 61/321,738, filed Apr. 7,2010; and PCT application number PCT/US2012/024169 filed Feb. 7, 2012,which claims the priority of U.S. provisional application No.61/440,260, filed Feb. 7, 2011; and U.S. provisional application No.61/451,455, filed Mar. 10, 2011, all of which are hereby incorporated byreference in their entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under grants numbered1RC1DK086378, RO1AI029549, P50DK064540 and RO1BK051406-12 each of whichwere awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention encompasses compounds and methods for treatingurinary tract infections.

BACKGROUND OF THE INVENTION

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli(UPEC) is one of the most common infectious diseases in women. Themorbidity and economic impact are enormous, with over $2.5 billion spentannually on treatment. Further, recurrent infections are a significantproblem despite appropriate antibiotic therapy of the index case. Thehigh rates of recurrence, and the large numbers of women that end up inurology clinics due to their chronic recurrent UTIs highlights the needfor a better understanding of the pathogenic mechanisms involved in thisdisease and the development of new and better therapies.

Gram-negative bacteria are the causative agents of a wide variety ofacute and chronic infectious diseases. Many of these infections areinitiated by a critical interaction between host ligands (frequentlypolysaccharide moieties) and bacterial adhesins (frequently expressed atthe distal tip of polymeric pilus fibers assembled by thechaperone/usher pathway). The mannose binding FimH adhesin of type 1pili is critical for the colonization and invasion into the bladderepithelium. After invasion, UPEC are able to rapidly multiply insidesuperficial umbrella cells of the bladder forming biofilm-likeintracellular bacterial communities (IBCs). Upon maturation, bacteriadisperse from the IBC, spread to neighboring cells, and form nextgeneration IBCs. This is the mechanism by which UPEC rapidly amplify innumbers in the urinary tract and cause disease.

The X-ray crystal structure of FimH bound to mannose showed that mannoseis bound in a negatively charged pocket on FimH. The mannose bindingsite is highly conserved as it is invariant in 300 fimH genes sequencedfrom clinical UPEC strains. Thus, FimH is the critical node of theentire UPEC pathogenic cascade.

Recurrence is a serious problem for many women. Women who present withan initial episode of acute UTI have a 25-44% chance of developing asecond and a 3% chance of experiencing three episodes within six monthsof the initial UTI. Recurrence occurs despite appropriate antibiotictreatment and clearance of the initial infection from the urine. A largepercentage of recurrent UTI are caused by the same strain of bacteria asthe initial infection. One study followed 58 women and found that 68% ofrecurrences were caused by the same initial index strain of UPEC asdetermined by restriction fragment length polymorphism (RFLP) analysis.In a separate study, 50% of recurrent strains isolated from femalecollege students appeared genotypically identical to the bacterialstrain corresponding to the initial UTI. Another long-term prospectivestudy demonstrated that the same strain of UPEC can cause a recurrentUTI up to 3 years later. The high frequency of same-strain recurrencessupports the notion that a UPEC reservoir can exist in the affectedindividual. The inventors have shown that a quiescent intracellularreservoir (QIR) can form in the bladder tissue itself after acuteinfection and persist even after antibiotic therapy and urine culturesbecome sterile. Thus, reactivation of bacteria in QIRs may also be acontributing factor in recurrent UTIs.

Therefore, there is a need for effective treatments that can cureurinary tract infections and prevent the formation of quiescentintracellular reservoir that are the source of so many recurrentinfections.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a compound comprisingformula (I):

wherein:

-   X is selected from the group consisting of hydrogen, OR², SR², and    NR^(z);-   Z is selected from the group consisting of O, S, CR³ and NR⁴;-   Y is selected from the group consisting of oxygen, sulfur, CR³, NR⁴,    —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—, —CH₂S—, CO,    —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—, —N(CH₂)_(n)—,    —(CH₂)_(n)—, NR⁵, and an optionally substituted alkyl, alkene,    alkyne, or heterocycle;-   R² is independently selected from the group consisting of hydrogen,    COR^(x), CONR^(x), hydrocarbyl, and substituted hydrocarbyl;-   R³, R⁴, R⁵ are independently selected from the group consisting of    hydrogen, hydrocarbyl, and substituted hydrocarbyl;-   R^(z) is independently selected from the group consisting of    hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),    —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);-   R^(x) is independently selected from the group consisting of    hydrogen, NR⁴R⁵, and an optionally substituted alkyl, cycloalkyl,    heterocycle, or aryl;-   n is an integer from 1 to 10; and-   R¹ is selected from the group comprising Formulas (IIIA), (IIIB),    (IV), (V), (VI), (VII) and (VIII):

wherein:

-   A is independently selected from the group consisting of CR⁶ and N;-   G is selected from the group consisting of S, O, CR⁸, and NR⁹;-   R⁶, R⁸ and R⁹ are independently selected from the group consisting    of hydrogen, hydrocarbyl, and substituted hydrocarbyl;-   R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently selected from the group    consisting of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵ COR¹⁶, —NR¹⁵CO₂R¹⁶,    —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen,    aryl, heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷, —SO₂NR¹⁸R¹⁹, alkenyl,    alkynyl, hydrocarbyl, and substituted hydrocarbyl;-   R¹⁵, R¹⁶, R¹⁷. R¹⁸, R¹⁹ are independently selected from the group    consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,    and heterocycle, and R¹⁶ and R¹⁷ together can optionally form a    ring, and R¹⁸ and R¹⁹ together can optionally form a ring; and-   a is either the integer 1 or the integer 2.

Another aspect of the invention encompasses a compound (II):

wherein

-   X is selected from the group consisting of hydrogen, OR², SR², and    NR^(z);-   Z is selected from the group consisting of O, S, CR³ and NR⁴;-   Y is selected from the group consisting of oxygen, sulfur, CR³, NR⁴,    —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—, —CH₂S—, CO,    —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—, —N(CH₂)_(n)—,    —(CH₂)_(n)—, NR⁵, and an optionally substituted alkyl, alkene,    alkyne, or heterocycle;-   L is selected from —O(CH₂)_(n)O—, —S(CH₂)_(n)S—, —N(CH₂)_(n)N—,    —(CH₂)_(n)—, —O[(CH₂)_(m)O(CH₂)_(n)]_(x)O—, —N(CH₂)_(m)O(CH₂)_(n)N—,    heterocycle, alkene, alkyne, —CON[(CH₂)_(m)O(CH₂)_(n)]_(x)NCO—,    —SO₂N[(CH₂)_(m)O(CH₂)_(n)]_(x)NO₂S—, and    NCO[(CH₂)_(m)O(CH₂)_(n)]_(x)CON—, where L is bound to a ring of R¹    at a meta or para position;-   R² is independently selected from the group consisting of hydrogen,    COR^(x), CONR^(x), hydrocarbyl, and substituted hydrocarbyl;-   R³, R⁴, R⁵ are independently selected from the group consisting of    hydrogen, hydrocarbyl, and substituted hydrocarbyl,-   R^(z) is independently selected from the group consisting of    hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),    —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);-   R^(x) is independently selected from the group consisting of    hydrogen, NR⁴R⁵, and an optionally substituted alkyl, cycloalkyl,    heterocycle, or aryl;-   m, n, and x are integers from 1 to 10; and-   R¹ is selected from the group comprising Formulas (IIIA), (IIIB),    (IV), (V), (VI), (VII) and (VIII):

wherein

-   A is independently selected from the group consisting of CR⁶ and N;-   G is selected from the group consisting of S, O, CR⁸, and NR⁹;-   R⁶, R⁸ and R⁹ are independently selected from the group consisting    of hydrogen, hydrocarbyl, and substituted hydrocarbyl;-   R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently selected from the group    consisting of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶,    —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen,    aryl, heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷, —SO₂NR¹⁸R¹⁹, alkenyl,    alkynyl, hydrocarbyl, and substituted hydrocarbyl;-   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the group    consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,    and heterocycle, and R¹⁶ and R¹⁷ together can optionally form a    ring, and R¹⁸ and R¹⁹ together can optionally form a ring; and-   a is either the integer 1 or the integer 2.

Yet another aspect of the present invention encompasses a method oftreating a urinary tract infection. The method comprises administering acompound of the invention to a subject in need thereof.

Still another aspect of the invention encompasses a method of reducingthe resistance of a bacterium to a bactericidal compound. The methodcomprises administering a compound of the invention to a subject in needthereof.

Other aspects and iterations of the invention are described below.

REFERENCE TO COLOR FIGURES

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts images and diagrams detailing uropathogenic E. colipathogenesis.

FIG. 2 depicts chaperone/usher mediated pilus biogenesis as shown forthe pilus system.

FIG. 3 depicts a GRASP view of the FimH adhesion domain bound to aD-mannose residue (shown in stick). Red=acidic residues. Blue=basicresidues.

FIG. 4 depicts a ribbon representation of the receptor binding pocket incomplex with the mannose. The D-mannose (pink), the mannose interactingresidues (green) and the residues of the hydrophobic ridge around thepocket (white) are shown as ball-and-stick model.

FIG. 5 depicts a graph showing the reduction of bacterial load in thebladder upon incubation with heptyl mannose at 6 hours post-infection.HM=heptyl mannose, MM=methyl mannose, p value obtained by Mann-Whitneytest.

FIG. 6 depicts FimH residues under positive selection. Green representsthe adhesin FimH and gray represents the chaperone FimC. Residues in redare under positive selection. Residues in blue are not under positiveselection. All residues are located outside the mannose binding pocket.

FIG. 7 depicts the alignment of FimH sequence from a panel of clinicalisolates previously examined. 18 clinical isolates examined for IBCformation were used to assess the different FimH sequences. The boldcolumns represent the residues identified as under positive selection.The residues highlighted in aqua represent residues that differ fromUTI89.

FIG. 8A,B depicts bacterial load and IBC formation of isolate acute4with its native FimH and UTI89's FimH. The FimH sequence from acute4 wasreplaced with the fimH sequence of UTI89 on the chromosome. (FIG. 8A)Depicts results obtained using bacterial titers, and (FIG. 8B) depictsresults obtained using IBC formation.

FIG. 9 depicts the structure of FimH with the compound number 15 inTable 1, illustrating interactions formed by compound number 15 withFimHA that are not shared with monomannose. Compound number 15 is shownin the final refined electron density calculated to 2.55 Å with 2Fo-Fccoefficients and contoured at 1σ.

FIG. 10 depicts a graph showing that mannosides number 7 and number 15(Table 1) reduced intracellular bacteria at 1 hour post-infection. A 1hour gentamicin protection assay was used to evaluate the amount ofbacteria present in the luminal versus intracellular fraction in thepresence of mannosides number 7 and number 15. A small decrease was seenin the luminal fraction in the presence of mannoside number 7 (p<0.05)whereas a significant decrease was seen in the intracellular fraction inthe presence of either mannoside (p<0.0001).

FIG. 11A,B depicts graphs showing that mannosides number 7 and number 15reduce infection capacity in vivo. (FIG. 11A) 6 hour LacZ revealssignificantly reduced IBCs at 1 mM and 0.1 mM mannoside for bothmannosides number 7 and number 15 (p<0.0001). (FIG. 11B) 6 hourbacterial load quantification reveals significantly reduced colonizationwith 1 mM mannoside number 7 and 1 mM mannoside number 15 treatedbacteria, p<0.001 and p<0.01, respectively.

FIG. 12 depicts a diagram showing pilicide inhibit bacterial pilusbiogenesis. The schematic diagram shows the model of chaperone-assistedpilus assembly and the mode of pilicide action. During normal pilusbiogenesis, newly synthesized pilus subunits cross into the periplasmvia the Sec secretion pathway. These pilus subunits bind to theirperiplasmic chaperone proteins (orange molecules) and fold into correctconfirmations (1). In the absence of the chaperones [A], pilus subunitsare degraded [B]. Chaperone-subunit complexes (2) are transported to theouter membrane usher proteins (blue cylinders) for assembly (3). Pilusassembly follows an ordered process with the chaperone-adhesin complexesas the initiatiors (4). As subunits are assembled into the growingpilus, accompanying chaperones disassociate from the membrane complexes(5), which could potentially be recycled to bind newly synthesizedsubunits. Pilicides (yellow circles) can cross freely through bacterialouter membranes and bind to periplasmic chaperones (7). Pilicides blockthe interaction of chaperone-subunit complexes with the outer membraneusher (8) and prevent pilus assembly (9 & 10).

FIG. 13A-C Effect of pilicides on pilus function and pilicideinteraction with the PapD chaperone. (FIG. 13A) E. coli NU14 was grownin the presence of pilicides 2d, 2a, 2b and 2c (np048) (3.5 mM each). HAtiters, adherence to 5637 bladder cells, and the ability to formbiofilms was determined. (FIG. 13B) New pilicides EC220-EC282 exhibitsignificant improvement in biofilm inhibitory activity compared to theoriginal pilicide, np048. (FIG. 13C) PapD-np048 complex in overlay withthe FimD1-125 N-terminal usher domain in complex with theFimC-FimH158-279 chaperone/adhesin complex. PapD and np048 are shown inlight blue ribbon and ball-and-stick representation, respectively. FimC,FimH158-279 and FimD1-125 N-terminal domain are shown in magenta, greenand yellow, respectively.

FIG. 14A,B depicts a graph showing that UTI89 bacteria, when inoculatedin the presence of FN075, makes significantly less IBCs in the bladder(FIG. 14A) and has a significantly reduced bacterial load within thebladder at 6 hours post-infection (FIG. 14B). p<0.0001.

FIG. 15 depicts a representation of an efficacy study in mice usingmannoside alone, antibiotic treatment alone, or dualmannoside/antibiotic treatment.

FIG. 16 depicts a graph showing that dual treatment of mice inoculatedwith bacteria showed significantly lower levels of bacteria in thebladder than antibiotics alone.

FIG. 17 depicts a diagram illustrating two strategies to inhibitbacterial binding to host cells. The schematic diagram shows the modelof chaperone-assisted pilus assembly and the mode of mannoside (leftpanel) and pilicide (right panel) action. During normal pilusbiogenesis, newly synthesized pilus subunits cross into the periplasmvia the Sec secretion pathway. These pilus subunits bind to theirperiplasmic chaperone proteins (orange molecules) and fold into correctconfirmations (1). In the absence of the chaperones, pilus subunits aredegraded (*). Chaperone-subunit complexes (2) are transported to theouter membrane usher proteins (blue cylinders) for assembly (3). Pilusassembly follows an ordered process with the chaperone-adhesin complexesas the initiators (4). As subunits are assembled into the growing pilus,accompanying chaperones disassociate from the membrane complexes (5),which could potentially be recycled to bind newly synthesized subunits.Mannosides (teal triangles) interact with the pilus adhesin and preventbinding to mannosylated residues (light brown triangles) on the surfaceof epithelial cells. Pilicides (yellow circles) can cross freely throughbacterial outer membranes and bind to periplasmic chaperones (B).Pilicides block the interaction of chaperone-subunit complexes with theouter membrane usher (C) and prevent pilus assembly (D).

FIG. 18A-C depicts diagrams illustrating chaperone-subunit donor strandexchange (DSE) and donor strand complementation (DSC). (FIG. 18A) Eachsubunit consists of six of the seven strands of a standardimmunoglobulin (Ig)-fold and an N-terminal extension (Nte). (FIG. 18B)In order to form a stable structure prior to incorporation into thegrowing fiber, the chaperone donates the missing seventh strand of thesubunit in a process called donor strand complementation (DSC). (FIG.18C) During pilus assembly, the free Nte of one subunit displaces thechaperone bound to another subunit and serves as the seventh strand ofthe Ig-like fold in a process called donor strand exchange (DSE).

FIG. 19A-E depicts the inhibition and disruption of UTI89 biofilm bymannoside and phase switching of UTI89 in the presence of mannoside.(FIG. 19A) Structure of mannosides. (FIG. 19B) The median inhibitoryconcentration (IC50) of mannoside on UTI89 biofilm formation. (FIG. 19C)The IC50 of mannoside on UTI89 biofilm disruption. After treatment withmannoside, the amount of UTI89 biofilm was measured. Bars show the meanvalue of the experiments (n=3). (FIG. 19D and FIG. 19E) ZFH-2056(Compound 50) dispersed biofilm as measured by confocal microscopy ofUTI89 biofilms grown for 24 h, then incubated for an additional 16 h inthe absence (FIG. 19D) or presence (FIG. 19E) of 0.3 μM ZFH-2056.

FIG. 20A-G depicts the mannoside effect on UTI89 colonization. (FIG.20A) Pretreatment of UTI89 with mannosides ZFH-2050 (Compound 49) andZFH-2056 (Compound 50) resulted in reduced IBC formation at 6 hpi. (FIG.20B) Urine PK analysis of ZFH-2056 (Compound 50) (n>3 mice) showingamounts in urine over time for each dosing regimen indicated. Horizontaldashed line is at IC50 (0.74 μM) as determined by the biofilm inhibitionassay. (c and d) Confocal microscopy of bladders from untreated (FIG.20C) and ZFH-2056-treated (FIG. 20D) mice. Bacteria were stained withSYTO9 (green) and the bladder luminal surface was stained with WGA(red). The image in c shows a normal, robust IBC whereas the arrows in dindicate luminal bacteria. (FIG. 20E) Total bacterial CFUs at 6 hpi fromuntreated mice or mice treated with ZFH-2056 either by IP (5 mg/kg) ororal (100 mg/kg) dosing 30 min prior to inoculation of UTI89. (FIG. 20F)IBC quantification at 6 hpi from untreated mice or mice treated withZFH-2056 either by IP (5 mg/kg) or oral (100 mg/kg) dosing 30 min priorto inoculation of UTI89. (FIG. 20G) 6 hpi ex vivo gentamicin protectionassay revealed both luminal and intracellular bacteria are significantlyreduced upon IP (5 mg/kg) pretreatment of mice with ZFH-2056. Barsindicate geometric mean. Statistical significance according toMann-Whitney is at **P<0.01, ***P<0.0001. ns, not significant; LOD,limit of detection.

FIG. 21A,B depicts mannoside synergy with TMP-SMZ at reducing UTI89colonization. (FIG. 21A) Total bacterial CFUs were quantitated 6 hpi.UTI89 colonization was reduced in mice treated with ZFH-2056 (Compound50), TMP-SMZ and TMP-SMZ+ZFH-2056. There was further decreasedcolonization in TMP-SMZ+ZFH-2056-treated mice over ZFH-2056 or TMP-SMZalone. PBC-1 colonization was reduced in mice treated with ZFH-2056 andTMP-SMZ+ZFH-2056, but not TMP-SMZ alone. Enhanced efficacy as measuredby bacterial CFUs was observed upon treatment with TMP-SMZ+ZFH-2056 overZFH-2056 or TMP-SMZ treatment alone. Bars indicate geometric mean.Statistical significance according to Mann-Whitney is at *P<0.05,**P<0.01, ***P<0.0001. ns, not significant; LOD, limit of detection.(FIG. 21B) Growth curve of PBC-1 with TMP-SMZ in the absence (solidline) or presence (dashed line) of 100 μM ZFH-2056. SMZ concentration is5×TMP concentration listed.

FIG. 22 depicts X-ray structure of biphenyl mannoside meta-methyl ester(Compound 29) with electron density (mesh) calculated with 2Fo-Fccoefficients, contoured at 1 sigma. Interaction with the Arg-98-Glu-50salt bridge (dashes), pi-pi stacking with Tyr-48 and hydrophobicinteraction with Tyr-137 are shown. Surface electrostatic potential ofFimH, calculated with APBS, is displayed such that pure blue and redwould be +4kT/e and −4kT/e respectively.

FIG. 23 depicts the correlation of HA Titer and Binding Data.

FIG. 24 depicts mannoside esters and their amidated counterparts withenhanced solubility.

FIG. 25A,B depicts results of hemagglutination (FIG. 25A) and LacZquantification of IBCs (FIG. 25B).

FIG. 26 depicts the pharmacokinetic results of ZFH56 (Compound 50)injected IP and PO into mice.

FIG. 27A-C depicts IBC numbers (FIG. 27A), bacterial titers (FIG. 27B)and lacZ staining (FIG. 27C) in mice treated with ZFH56 (Compound 50).

FIG. 28A-C depicts fluorescent microscopy imaging of infected bladderstreated with ZFH56 (Compound 50).

FIG. 29 depicts efficacy studies of mannosides against TMP-SMZ resistantstrains.

FIG. 30A-C depicts efficacy of ZFH56 (Compound 50) under various dosesof inoculum.

FIG. 31 depicts efficacy studies of ZFH56 (Compound 50) with compounddose boosting.

FIG. 32A-B depicts time course of infection and treatment withmannosides (FIG. 32A) and inhibition of preformed biofilm by ZFH56(Compound 50) (FIG. 32B).

FIG. 33 depicts a graph showing that there is no defect in UTI189invasion following implantation. Graph represents bacterial titers fromhomogenized bladders from non-implanted (∘) or implanted (●) animalsinfected with UTI189 3 hpi following gentamicin protection assay.Horizontal dashed lines represent the limit of detection (lod) forviable bacteria (Int=intracellular, Ext=Extracellular). Each symbolrepresents a mouse from two independent experiments with n=5/condition.The horizontal bars represent the median of each dataset; p value by theMann Whitney U test.

FIG. 34A-C depicts microscope images and a graph showing thaturopathogenic E. coli produce IBCs in the superficial umbrella cells ofimplanted bladders. (FIG. 34A) Representative images of splayed bladdersof female C57Bl/6Ncr mice infected with UTI189 6 hpi in the absence(non-implanted) or presence (Implanted) of implants following LacZstaining. Each black arrow indicates a purple speck, indicative of anIBC. (FIG. 34B) Quantification of IBC formation following LacZ stainingat 6 hpi. Each symbol represents IBC number from a single mouse from twoindependent experiments with n=5/group. p value obtained from the MannWhitney U test. (FIG. 34C) Representative CLSM images of whole bladdersfrom non-implanted and implanted animals infected with UTI189ectopically expressing GFP (Green), stained with DNA dye SYTO83 (Red)and Alexa-fluor 633-conjugate of WGA (Blue) reveal the presence of IBCwithin umbrella cells. Scale bar=20 μm.

FIG. 35A-F depicts micrographs showing that IBC and filamentation occurfollowing urinary catheterization. Representative CLSM images of wholebladders from non-implanted and implanted animals infected with UTI189ectopically expressing GFP (Green), stained with DNA dye SYTO083 (Red)and Alexa-fluor 633-conjugate of WGA (Blue) reveal the presence ofmultiple IBCs within single umbrella cells (FIG. 35A-B), that unlikenon-implanted bladders (FIG. 35C), the underlying epithelium is exposedfollowing urinary catheterization (FIG. 35D-F), depict the absence ofbacterial colonization of the exposed underlying epithelium in implantedanimals (FIGS. 35D-F), and the presence of filamenting bacteria inumbrella cells (FIGS. 35E-F). Scale bar=20 μm

FIG. 36A-B depicts two graphs showing that UPEC reservoir reactivationcan lead to urinary implant and bladder colonization. Graphs representbacterial titers in log scale recovered from implants, homogenizedbladders and kidneys of non-bacteriuric animals 14 days post infectionwith UTI89HK::GFP that were non-implanted or implanted for 3 day (FIG.36A) or 5 days (FIG. 36B). Horizontal dashed lines represent the limitof detection for viable bacteria. Each symbol represents a mouse from atleast two independent experiments with n=10-20/group/experiment. Thehorizontal bars represent the median of each dataset; p value by theMann Whitney U test.

FIG. 37A-C depicts two graphs showing that deletion of fimH reducesbiofilm formation and attenuates UPEC virulence. Graphs representcrystal violet based quantification (FIG. 37A) and CFU enumeration inlogarithmic scale (log scale) (FIG. 37B) of 24 h old UTI89 andUTI89ΔfimH (ΔfimH) biofilms under human urine flow on silicone tubingsat 37° C. indicating that ΔfimH is defective in biofilm formation underthese conditions. Bars represent mean of three independent experiments,error bars indicate standard error of the mean (SEM). p values from MannWhitney U test. (FIG. 37C) Graph represents bacterial titers in logscale recovered from implants, homogenized bladders and kidneys ofnon-implanted (open symbols) and implanted (closed symbols) infectedwith either UTI89 (square) or ΔfimH (circle) for 24 h. Horizontal dashedlines represent the limit of detection for viable bacteria. Each symbolrepresents a mouse from at least two independent experiments withn=5/group. The horizontal bars represent the median of each dataset;*p<0.05 and ***p<0.0005 by the Mann Whitney U test.

FIG. 38A-B depicts two graphs showing that S pili and curli are notcritical for UPEC virulence following urinary catheterization. Graphsrepresent bacterial titers in log scale recovered at 24 hpi fromimplants, homogenized bladders and kidneys of (FIG. 38A) implantedanimals infected with either UTI189 (*) or UTI89 mutant strainsdeficient in type 1 pili, ΔfimH (▪), S pili ΔsfaA-H(▴), both type 1 andS pili ΔsfaA-HΔfimB-H(♦) and (FIG. 38B) non-implanted (open symbols) orimplanted (closed symbols) animals infected with UTI89 (∘,▪), ΔfimH (□,▪) or UTI89 mutant strains deficient in curli components ΔcsgA (Δ, ▴)and ΔcsgBΔcsgG (♦,⋄). Horizontal dashed lines represent the limit ofdetection for viable bacteria. Each symbol represents a mouse from atleast two independent experiments with n=5/group. The horizontal barsrepresent the median of each dataset; *p<0.05, ***p<0.005 **p<0.0005, nscorresponds to p>0.05 by the Mann Whitney U test.

FIG. 39A-B depicts two graphs showing that methyl mannose inhibits UPECbiofilm in human urine. Graphs represent crystal violet basedquantification (FIG. 39A) and CFU enumeration in logarithmic scale (logscale) (FIG. 39B) of 24 h old UTI89 biofilms in human urine with orwithout 1% methyl mannose under flow on silicone tubings at 37° C.indicating that methyl mannose prevents UPEC biofilm formation. Barsrepresent mean of three independent experiments, error bars indicatestandard error of the mean (SEM). p values from Mann Whitney U test.

FIG. 40A-B depicts two graphs showing that mannoside treatment preventsIBC formation and UPEC virulence when used in combination with TMP-SMZ.(FIG. 40A) Graph represents IBC enumeration from LacZ staining ofsplayed bladders of female C57Bl/6Ncr mice treated i.p. with mannosideor saline prior to transurethral implantation and inoculation with UTI896 hpi. Each symbol represents IBC number from a single mouse from twoindependent experiments with n=5/group. p value obtained from the MannWhitney U test. (FIG. 40B) Graph represents bacterial titers in logscale recovered at 6 hpi from implants, homogenized bladders and kidneysof animals treated with saline (∘), mannoside (□), TMP-SMZ (●) andTMP-SMZ+Mannoside (▪) prior to urinary implantation and inoculation withUTI89. Horizontal dashed lines represent the limit of detection forviable bacteria. Each symbol represents a mouse from at least twoindependent experiments with n=5/group. The horizontal bars representthe median of each dataset; *p<0.05, **p<0.0005, ***p<0.0005, nscorresponds to p>0.05 by the Mann Whitney U test.

FIG. 41 depicts a graph showing that treatment of chronic mice with 5doses of Mannoside 8 eliminates UPEC from the bladder. Mannoside 8treatment (▴), PBS treatment (●).

FIG. 42 depict a graph showing that Mannoside 8 is effective against themultidrug resistant UPEC isolate EC958. Mannoside 8 treatment (Δ), PBStreatment (▴).

FIG. 43A-E depicts inhibition, prevention and disruption of UTI89biofilm by mannoside. (FIG. 43A), Discovery of biphenyl mannoside leadFimH inhibitors. Cellular HAI titers (EC_(>90)) are shown inparentheses. (FIG. 43B) The median inhibitory concentration (IC₅₀) ofmannosides 1-3 and 6 on UTI89 biofilm formation. Mannoside was added atthe initiation of biofilm formation. (FIG. 43C) The IC50 of 1-3 and 6 onUTI89 biofilm prevention. Mannoside was added 24 h after biofilm growthwas initiated and % biofilm was calculated 16 h after addition ofmannoside. Bars show the mean value of the experiments (n=3). (FIG. 43D,E) 6 dispersed biofilm as measured by confocal microscopy of UTI89biofilms grown for 24 h (FIG. 43D), then incubated for an additional 16h in the presence of 0.3 μM 6 (FIG. 43E).

FIG. 44A-G depicts mannoside effect on UTI89 colonization. (FIG. 44A)Urine PK analysis of 6 (n≥3 mice) showing amounts in urine over time foreach dosing regimen indicated. Horizontal dashed line is at IC₅₀ (0.74μM) as determined by the biofilm inhibition assay. (FIG. 44B) Mannosideeffectively treats UTI. Chronically infected mice were treated with PBSor 6 (PO, 100 and 50 mg/kg). 6 hours post-treatment bacterial counts inthe bladder were enumerated. In the mannoside-treated groups, there wasa significant 3-log drop in bacterial load relative to PBS-treated mice.(C, D) Confocal microscopy of bladders from PBS-treated (FIG. 44C) and6-treated (FIG. 44D) mice. Bacteria were stained with SYTO9 (green) andthe bladder luminal surface was stained with WGA (red). The image in Cshows a normal, robust IBC whereas the arrows in D indicate luminalbacteria. (FIG. 44E) Total bacterial CFUs at 6 hpi from mice treatedwith PBS or 6 either by IP (5 mg/kg) or oral (100 mg/kg) dosing 30 minprior to inoculation of UTI89. (F) IBC quantification at 6 hpi from micetreated with PBS or 6 either by IP (5 mg/kg) or oral (100 mg/kg) dosing30 min prior to inoculation of UTI89. (FIG. 44G) 6 hpi ex vivogentamicin protection assay revealed both luminal and intracellularbacteria are significantly reduced upon IP (5 mg/kg) pretreatment ofmice with 6. Bars indicate geometric mean. Statistical significanceaccording to Mann-Whitney is at *P<0.05, **P<0.01, ***P<0.0001. ns, notsignificant; LOD, limit of detection.

FIG. 45 depicts mannoside potentiates TMP-SMZ treatment. Total bacterialCFUs were quantified 6 hpi. UTI89 colonization was reduced in micetreated with 6, TMP-SMZ and TMP-SMZ+6. There was further decreasedcolonization in TMP-SMZ+6-treated mice over 6 or TMP-SMZ alone. PBC-1colonization was reduced in mice treated with 6 and TMP-SMZ+6, but notTMP-SMZ alone. Enhanced efficacy as measured by bacterial CFUs wasobserved upon treatment with TMP-SMZ+6 over 6 or TMP-SMZ treatmentalone. Bars indicate geometric mean. Statistical significance accordingto Mann-Whitney is at *P<0.05, **P<0.01, ***P<0.0001. ns, notsignificant; LOD, limit of detection.

FIG. 46A-C depicts newly designed mannosides show enhanced PK andpotency at treating infection. (FIG. 46A) Optimized ortho-methyl andtrifluoromethyl substituted biphenyl mannosides. Cellular HAI titers(EC_(>90)) are shown in parentheses. (FIG. 46B) Mannosides show improvedPK. Mannosides 8 and 10 dosed at 50 mg/kg had equivalent levels ofcompound in the urine 6 h post-treatment relative to mannoside 6 dosedat 100 mg/kg. (FIG. 46C) Chronically infected mice were treated with PBSor mannoside 6, 10 or 8 (PO, 50 mg/kg). 6 hours posttreatment bacterialcounts in the bladder were enumerated. In the mannoside-treated groups,there was a significant 3-log drop in bacterial load relative toPBS-treated mice. The optimized mannoside 8 showed increased efficacyover 6.

FIG. 47 depicts structures of substituted biphenyl mannosides.

FIG. 48 depicts a graph showing curves of HAI, Octet and DSF assayresults.

FIG. 49 depicts a proposed model of mannoside 7 bound to FimH calculatedwith APBS (Adaptive Poisson-Boltzmann Solver) software using PDB code:3MCY.

FIG. 50A-B depicts (FIG. 50A) Metabolic lability of the mannoside 3glycosidic bond from hydrolysis to mannose and biphenol; (FIG. 50B)Elimination kinetics and clearance of mannoside 3 and biphenol (R)hydrolysis product in mouse urine.

FIG. 51A depicts plasma pharmacokinetics of optimized ortho-substitutedmannosides 5a-c, 7, 8 and mannoside 3. (FIG. 51B) depicts eliminationclearance kinetics of optimized ortho-substituted mannosides 5a-c, 7, 8and mannoside 3 in urine.

FIG. 52A-B depicts two graphs showing that UTI89ΔqseC has a defect inbladder invasion. (FIG. 52A) Bladder titers at 1 and 3 h.p.i showingdecreased overall bacterial numbers recovered from bladders infectedwith UTI89ΔqseC compared to those infected with wt UTI89. (FIG. 52B)Bacterial titers recovered from gentamicin-treated bladders(intracellular population) after washing 3 times with PBS to remove theextracellular, adherent bacteria (luminal population). Fewer UTI89ΔqseCbacteria are internalized, indicating that the qseC mutant is lessefficient in invading the host bladder compared to wt UTI89. The averageof 3 independent experiments is shown (*, P<0.02; **, P<0.003, bytwo-tailed Mann-Whitney).

FIG. 53A-D depicts a diagram detailing and data showing that restoringproduction of type 1 pili in UTI89ΔqseC does not influence otherΔqseC-mediated defects. (FIG. 53A) Schematic showing the strategy usedto lock the phase-variable fim promoter in the ON orientation. (FIG.53B) Hemagglutination assays and (FIG. 53C) western blot analysesdepicting restoration of type 1 pili expression in UTI89ΔqseC locked ONstrain (ΔqseC_LON). (FIG. 53D) Assessment of curli production onYESCA-CR agar verifies that, similarly to UTI89ΔqseC, UTI89ΔqseC_LONremains defective for curli expression, exhibiting a white and smoothmorphotype. In contrast, wt UTI89 and UTI89_LON appear red, dry andrough a phenotype indicative of curli expression.

FIG. 54 depicts a graph showing co-inhibition of type 1 pili and QseC asa prophylactic measure for UTIs. Chart depicting the bladder cfuobtained at 2 wks post infection from mice pre-treated with mannosideand subsequently infected with either wt UTI89, or UTI89ΔqseC (used as aproxy for a QseC inhibitor). Significantly fewer cfu (1.5-log reduction)were obtained from pre-treated mice infected with UTI89ΔqseC. Theaverage of 3 independent experiments is shown (**, P<0.01; ***, P<0.001,by two-tailed Mann-Whitney). UTI89_MAN, mice pre-treated with mannosideand challenged with wt UTI89; ΔqseC_MAN, mice pre-treated with mannosideand challenged with UTI89ΔqseC; UTI89, mice pre-treated with PBS andchallenged with wt UTI89; ΔqseC, mice pre-treated with PBS andchallenged with UTI89ΔqseC.

DETAILED DESCRIPTION

Compounds that inhibit the function of type I pili of bacteria have beendeveloped. The compounds may be useful for the treatment of urinarytract infections. Significantly, the compounds may prevent bacterialcolonization and invasion of the bladder tissue to prevent infection andthe establishment of reservoirs that can serve as a source of recurrentinfections. The invention also encompasses methods of use of a compoundof the invention.

I. Compounds

One aspect of the invention is a compound of Formula (I):

wherein:

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        and NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R² is independently selected from the group consisting of        hydrogen, COR^(x), CONR^(x), hydrocarbyl, and substituted        hydrocarbyl;    -   R³, R⁴, R⁵ are independently selected from the group consisting        of hydrogen, hydrocarbyl, and substituted hydrocarbyl;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl;    -   n is an integer from 1 to 10; and    -   R¹ is selected from the group comprising Formulas (IIIA),        (IIIB), (IV), (V), (VI), (VII) and (VIII):

wherein:

-   -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently selected from the        group consisting of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶,        —NR¹⁵CO₂R¹⁶, —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro,        cyano, halogen, aryl, heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷,        —SO₂NR¹⁸R¹⁹, alkenyl, alkynyl, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷. R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring; and a is either the integer 1 or the integer 2.

One embodiment of formula (I), where R¹ is formula (IIIA), comprisesformula (IX):

wherein:

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R², R³, R⁴, R⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹⁰ is independently selected from the group consisting of        hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶, —NR¹⁵CONR¹⁶,        —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen, aryl,        heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷, —SO₂NR¹⁸R¹⁹, alkenyl, alkynyl,        hydrocarbyl, and substituted hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl; and    -   a is either the integer 1 or the integer 2.

In an exemplary embodiment of a compound of formula (IX), R¹⁰ is aryl orheterocyle.

Another embodiment of formula (I), where R¹ is formula (IIIB), comprisesformula (X):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        and NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R² is independently selected from the group consisting of        hydrogen, —COR^(x), and —CONR^(x);    -   R³, R⁴, R⁵ are independently selected from the group consisting        of hydrogen, hydrocarbyl, and substituted hydrocarbyl;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹⁰ is independently selected from the group consisting of        hydrogen, OR¹⁵, NR¹⁵R¹⁶, NR¹⁵COR¹⁶, NR¹⁵CO₂R¹⁶, NR¹⁵CONR¹⁶,        NR¹⁵SO₂R¹⁶, COR¹⁵, SO₂R¹⁵, nitro, cyano, halogen, aryl,        heterocycle, COOR¹⁵, CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹, alkenyl, alkynyl,        hydrocarbyl, and substituted hydrocarbyl; and    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring; and a is either the integer 1 or the integer 2.

Yet another embodiment of formula (I), where R¹ is formula (IV),comprises formula (XI):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R², R³, R⁴, R⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹¹ and R¹² are independently selected from the group consisting        of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶,        —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano,        halogen, aryl, heterocycle, COOR¹⁵, CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹,        alkenyl, alkynyl, hydrocarbyl, and substituted hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl; and    -   a is either the integer 1 or the integer 2.

Still another embodiment of formula (I), where R¹ is formula (V),comprises formula (XII):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R², R³, R⁴, R⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10    -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO²R¹⁶,        —NR¹⁵CONR¹⁶, NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen,        aryl, heterocycle, COOR¹⁵, CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹, alkenyl,        alkynyl, hydrocarbyl, and substituted hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl; and    -   a is either the integer 1 or the integer 2.

Yet still another embodiment of formula (I), where R¹ is formula (VI),comprises formula (XIII):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R², R³, R⁴, R⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   R⁶ is independently selected from the group consisting of        hydrogen, hydrocarbyl, and substituted hydrocarbyl;    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶,        —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano,        halogen, aryl, heterocycle, COOR¹⁵, CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹,        alkenyl, alkynyl, hydrocarbyl, and substituted hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl; and    -   a is either the integer 1 or the integer 2.

Still another embodiment of formula (I), where R¹ is formula (VII),comprises formula (XIV):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R², R³, R⁴, R⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   R⁶ is independently selected from the group consisting of        hydrogen, hydrocarbyl, and substituted hydrocarbyl;    -   R¹³ and R¹⁴ are independently selected from the group consisting        of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶,        —NR¹⁵CONR¹⁶, —NR¹⁵SO2R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano,        halogen, aryl, heterocycle, COOR¹⁵, CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹,        alkenyl, alkynyl, hydrocarbyl, and substituted hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl; and    -   a is either the integer 1 or the integer 2.

A further embodiment of formula (I), where R¹ is formula (VIII),comprises formula (XV):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        and NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   R², R³, R⁴, R⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹¹ and R¹² are independently selected from the group consisting        of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶,        —NR¹⁵CONR¹⁶, NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen,        aryl, heterocycle, COOR¹⁵, CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹, alkenyl,        alkynyl, hydrocarbyl, and substituted hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring;    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, —NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl; and    -   a is either the integer 1 or the integer 2.

A still further another embodiment of formula (I), where R¹ is formula(IV), comprises formula (XVI):

wherein

-   -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of O, S, CR³, and NR⁴;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   R³, R⁴, and R⁶ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹¹ is independently selected from the group consisting of        hydrogen, —NR⁵, —OR⁵, nitro, cyano, chloro, bromo, iodo, fluoro,        —COOR¹⁵, —CONR¹⁶R¹⁷, —SO₂R¹⁵, —SO₂NR¹⁸R¹⁹, methyl, hydrocarbyl,        and substituted hydrocarbyl;    -   R¹² is independently selected from the group consisting of        hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶, —NR¹⁵CONR¹⁶,        —NR¹⁵SO₂R¹⁶, COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen, aryl,        heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹, hydrocarbyl, and        substituted hydrocarbyl; and    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring; and    -   a is either the integer 1 or the integer 2.

A still further another embodiment of formula (I), where R¹ is formula(IV), comprises formula (XVII):

wherein

-   -   Y is selected from the group consisting of O, S, CR³, and NR⁴;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   R³, R⁴, and R⁶ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹¹ is selected from the group consisting of hydrogen, —NR⁵,        —OR⁵, nitro, cyano, chloro, bromo, iodo, fluoro, —COOR¹⁵,        —CONR¹⁶R¹⁷, —SO₂R¹⁵, —SO₂NR¹⁸R¹⁹, methyl, hydrocarbyl, and        substituted hydrocarbyl;    -   R¹² is selected from the group consisting of hydrogen, —OR¹⁵,        —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR₁₅CO₂R¹⁶, —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶,        —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen, aryl, heterocycle,        —COOR¹⁵, —CONR¹⁶R¹⁷, —SO₂NR¹⁸R¹⁹, hydrocarbyl, and substituted        hydrocarbyl;    -   R²⁰ is selected from the group consisting of —NR¹⁵SO₂R¹⁶,        —COOR¹⁵, —NR¹⁵CONR¹⁶, —CONR¹⁶R¹⁷, —SO₂NR¹⁸R¹⁹; and    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring.

In a preferred embodiment of formula (XVII), R¹¹ is selected from thegroup consisting of chloro, bromo, iodo, and fluoro, R¹² is selectedfrom the group consisting of nitro, cyano, —COOR¹⁵, —CONR¹⁶R¹⁷, and—SO₂NR¹⁸R¹⁹, and R²⁰ is selected from the group consisting of —COOR¹⁵,—NR¹⁵CONR¹⁶, —CONR¹⁶R¹⁷, and —SO₂NR¹⁸R¹⁹.

In another preferred embodiment of formula (XVII), R¹¹ is chloro, R¹² isselected from the group consisting of nitro, cyano, —COOR¹⁵, —CONR¹⁶R¹⁷,and —SO₂NR¹⁸R¹⁹, and R²⁰ is selected from the group consisting of—COOR¹⁵, —NR¹⁵CONR¹⁶, —CONR¹⁶R¹⁷, and —SO₂NR¹⁸R¹⁹.

A yet further embodiment of formula (I), where R¹ is formula (VIII),comprises formula (XVIII):

wherein

-   -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of O, S, CR³, and NR⁴;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R³, R⁴, R⁶, R⁸, and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹¹ is independently selected from the group consisting of        hydrogen, —NR⁵, —OR⁵, nitro, cyano, chloro, bromo, iodo, fluoro,        —COOR¹⁵, —CONR¹⁶R¹⁷, —SO₂R¹⁵, —SO₂NR¹⁸R¹⁹, methyl, hydrocarbyl,        and substituted hydrocarbyl;    -   R¹² is independently selected from the group consisting of        hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶, —NR¹⁵CONR¹⁶,        —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen, aryl,        heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹, hydrocarbyl, and        substituted hydrocarbyl; and    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring; and    -   a is either the integer 1 or the integer 2.

In some embodiments of formula (I), where R¹ is formula (VIII),comprises formula (XIX):

wherein

-   -   Y is selected from the group consisting of O, S, CR³, and NR⁴;    -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R³, R⁴, R⁶, R⁸, and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹¹ is independently selected from the group consisting of        hydrogen, —NR⁵, —OR⁵, nitro, cyano, chloro, bromo, iodo, fluoro,        —COOR¹⁵, —CONR¹⁶R¹⁷, SO₂R¹⁵, —SO₂NR¹⁸R¹⁹, methyl, hydrocarbyl,        and substituted hydrocarbyl;    -   R¹² is independently selected from the group consisting of        hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶, —NR¹⁵CO₂R¹⁶, —NR¹⁵CONR¹⁶,        —NR¹⁵SO₂R¹⁶, COR¹⁵, —SO₂R¹⁵, nitro, cyano, halogen, aryl,        heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷, SO₂NR¹⁸R¹⁹, hydrocarbyl, and        substituted hydrocarbyl; and    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring.

In a preferred embodiment of formula (XIX), R¹¹ is selected from thegroup consisting of chloro, bromo, iodo, and fluoro, R¹² is selectedfrom the group consisting of —COOR¹⁵, and —CONR¹⁶R¹⁷, and R²⁰ isselected from the group consisting of —COOR¹⁵, —NR¹⁵CONR¹⁶, —CONR¹⁶R¹⁷,and —SO₂NR¹⁸R¹⁹.

In another preferred embodiment of formula (XVII), R¹¹ is chloro, R¹² isselected from the group consisting of —COOR¹⁵, and —CONR¹⁶R¹⁷, and R²⁰is selected from the group consisting of —COOR¹⁵, —NR¹⁵CONR¹⁶,—CONR¹⁶R¹⁷, and —SO₂NR¹⁸R¹⁹.

Another aspect of the present invention is a compound of formula (II):

wherein

-   -   X is selected from the group consisting of hydrogen, OR², SR²,        and NR^(z);    -   Z is selected from the group consisting of O, S, CR³ and NR⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CR³,        NR⁴, —N(R⁵)CO—, —CH₂N(R⁵)—, —CH₂N(R⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(R⁵)—, —SO₂N(R⁵)—, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, NR⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   L is selected from —O(CH₂)_(n)O—, —S(CH₂)_(n)S—, —N(CH₂)_(n)N—,        —(CH₂)_(n)—, —O[(CH₂)_(m)O(CH₂)_(n)]_(x)O—,        —N(CH₂)_(m)O(CH₂)_(n)N—, heterocycle, alkene, alkyne,        —CON[(CH₂)_(m)O(CH₂)_(n)]_(x)NCO—,        —SO₂N[(CH₂)_(m)O(CH₂)_(n)]_(x)NO₂S—, and        NCO[(CH₂)_(m)O(CH₂)_(n)]_(x)CON—, where L is bound to a ring of        R¹ at a meta or para position;    -   R² is independently selected from the group consisting of        hydrogen, COR^(x), CONR^(x), hydrocarbyl, and substituted        hydrocarbyl;    -   R³, R⁴, R⁵ are independently selected from the group consisting        of hydrogen, hydrocarbyl, and substituted hydrocarbyl,    -   R^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COR^(x),        —CONR^(x)R^(x)SO₂R^(x), and —CO₂R^(x);    -   R^(x) is independently selected from the group consisting of        hydrogen, NR⁴R⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl;    -   m, n, and x are integers from 1 to 10; and    -   R¹ is selected from the group comprising Formulas (IIIA),        (IIIB), (IV), (V), (VI), (VII) and (VIII):

wherein

-   -   A is independently selected from the group consisting of CR⁶ and        N;    -   G is selected from the group consisting of S, O, CR⁸, and NR⁹;    -   R⁶, R⁸ and R⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently selected from the        group consisting of hydrogen, —OR¹⁵, —NR¹⁵R¹⁶, —NR¹⁵COR¹⁶,        —NR¹⁵CO₂R¹⁶, —NR¹⁵CONR¹⁶, —NR¹⁵SO₂R¹⁶, —COR¹⁵, —SO₂R¹⁵, nitro,        cyano, halogen, aryl, heterocycle, —COOR¹⁵, —CONR¹⁶R¹⁷,        —SO₂NR¹⁸R¹⁹, alkenyl, alkynyl, hydrocarbyl, and substituted        hydrocarbyl;    -   R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ are independently selected from the        group consisting of hydrogen, hydrocarbyl, substituted        hydrocarbyl, aryl, and heterocycle, and R¹⁶ and R¹⁷ together can        optionally form a ring, and R¹⁸ and R¹⁹ together can optionally        form a ring; and    -   a is either the integer 1 or the integer 2.

In an exemplary alternative of each of the foregoing embodiments, acompound comprising formula (I) is a compound comprising any of theFormulas in Table 15.

In a further exemplary alternative of each of the foregoing embodiments,a compound comprising formula (I) is compound number 50, 75, 76, or 77.

Another aspect of the invention is a compound of Formula (XXI):

wherein

-   -   X is selected from the group consisting of hydrogen, OD², SD²,        ND^(z);    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is oxygen;    -   D², D³, and D⁴ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D⁵ is selected from the group consisting of CF₃, halogen, CH₃,        OMe, hydrocarbyl, and substituted hydrocarbyl;    -   D⁶, D⁷, D⁸, and D⁹ are independently selected from the group        consisting of hydrogen, —COD¹⁰D¹¹, —COND¹⁰D¹¹, —COOD¹², and        —ND¹²COND¹⁰, or D⁶ and D⁷ may optionally form a cycloalkyl or        heterocyclo ring, D⁷ and D⁸ may optionally form a cycloalkyl or        heterocyclo ring, and D⁸ and D⁹ may optionally form a cycloalkyl        or heterocyclo ring; and    -   D¹⁰, D¹¹, and D¹² are independently selected from the group        consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,        aryl, and heterocycle;    -   D¹⁸ and D¹⁹ are independently selected from the group consisting        of hydrogen, hydrocarbyl, and substituted hydrocarbyl;    -   D^(z) is independently selected from the group consisting of        hydrogen, hydrocarbyl, substituted hydrocarbyl, —COD^(x),        —COND^(x)D^(x)SO₂D^(x), and —CO₂D^(x); and    -   D^(x) is independently selected from the group consisting of        hydrogen, —ND¹⁸D¹⁹, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl.

In one embodiment, a compound of the invention comprises Formula (XXI),wherein

-   -   X is selected from the group consisting of OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is oxygen;    -   D², D³, and D⁴ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D⁵ is selected from the group consisting of CF₃, halogen, CH₃,        and OMe;    -   D⁶, D⁷, D⁸, and D⁹ are independently selected from the group        consisting of hydrogen, —COD¹⁰D¹¹, —COND¹⁰D¹¹, —COOD¹², and        —ND¹²COND¹⁰, or D⁶ and D⁷ may optionally form a cycloalkyl or        heterocyclo ring, D⁷ and D⁸ may optionally form a cycloalkyl or        heterocyclo ring, and D⁸ and D⁹ may optionally form a cycloalkyl        or heterocyclo ring; and    -   D¹⁰, D¹¹, and D¹² are independently selected from the group        consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,        aryl, and heterocycle.

In another embodiment, a compound of the invention comprises Formula(XXI), wherein

-   -   X is selected from the group consisting of OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is oxygen;    -   D², D³, and D⁴ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D⁵ is selected from the group consisting of CF₃, halogen, CH₃,        and OMe;    -   D⁶ and D⁸ are hydrogen;    -   D⁷ and D⁹ are independently selected from the group consisting        of hydrogen, —COD¹¹D¹¹, —COND¹⁰D¹¹, —COOD¹², and —ND¹²COND¹⁰;        and    -   D¹⁰, D¹¹, and D¹² are independently selected from the group        consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,        aryl, and heterocycle.

In yet another embodiment, a compound of the invention comprises Formula(XXI), wherein

-   -   X is OH;    -   Z is O;    -   Y is oxygen;    -   D², D³, and D⁴ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D⁵ is selected from the group consisting of CF₃, halogen, CH₃,        and OMe;    -   D⁶ and D⁸ are hydrogen;    -   D⁷ and D⁹ are independently selected from the group consisting        of hydrogen, —CONHCH₃, —COOCH₃, and —NHCONH₂.

Another aspect of the invention is a compound of Formula (XXII):

wherein

-   -   X is selected from the group consisting of hydrogen, OD², SD²,        ND^(z);    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of sulfur, CD³, ND⁴,        —N(D¹⁸)CO—, —CH₂N(D¹⁸)-, —CH₂N(D¹⁸)CO—, —CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D¹⁸)-, —SO₂N(D¹⁸)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, ND¹⁸, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   n is an integer from 1 to 10;    -   D², D³, and D⁴ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D¹⁰ is selected from the group consisting of hydrogen, CF₃,        halogen, CH₃, OMe, hydrocarbyl, and substituted hydrocarbyl;    -   D¹¹, D¹², D¹³, and D¹⁴ are independently selected from the group        consisting of hydrogen, —COD¹⁵D¹⁶, —COND¹⁵D¹⁶, —COOD¹⁷, and        —ND¹⁷COND¹⁵, or D¹¹ and D¹² may optionally form a cycloalkyl or        heterocyclo ring, D¹² and D¹³ may optionally form a cycloalkyl        or heterocyclo ring, and D¹³ and D¹⁴ may optionally form a        cycloalkyl or heterocyclo ring;    -   D¹⁵, D¹⁶, and D¹⁷ are independently selected from the group        consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,        aryl, and heterocycle;    -   D¹⁸ and D¹⁹ are independently selected from the group consisting        of hydrogen, hydrocarbyl, and substituted hydrocarbyl; and    -   D^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COD^(x),        —COND^(x)D^(x)SO₂D^(x), and —CO₂D^(x); and    -   D^(x) is independently selected from the group consisting of        hydrogen, —ND¹⁸D¹⁹, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl.

In one embodiment, a compound of the invention comprises Formula (XXII),wherein

-   -   X is OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of sulfur, CD³, ND⁴,        —N(D¹⁸)CO—, —CH₂N(D¹⁸)-, —CH₂N(D¹⁸)CO—, —CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D¹⁸)-, —SO₂N(D¹⁸)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, ND¹⁸, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   n is an integer from 1 to 10;    -   D², D³, D⁴ and D¹⁸ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D¹⁰ is selected from the group consisting of hydrogen, CF₃,        halogen, CH₃, and OMe;    -   D¹¹, D¹², D¹³, and D¹⁴ are independently selected from the group        consisting of hydrogen, —COD¹⁵D¹⁶, —COND¹⁵D¹⁶, —COOD¹⁷, and        —ND¹⁷COND¹⁵, or D¹¹ and D¹² may optionally form a cycloalkyl or        heterocyclo ring, D¹² and D¹³ may optionally form a cycloalkyl        or heterocyclo ring, and D¹³ and D¹⁴ may optionally form a        cycloalkyl or heterocyclo ring; and    -   D¹⁵, D¹⁶, and D¹⁷ are independently selected from the group        consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,        aryl, and heterocycle.

In another embodiment, a compound of the invention comprises Formula(XXII), wherein

-   -   X is OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of sulfur, CD³, ND⁴,        —N(D¹⁸)CO—, —CH₂N(D¹⁸)-, —CH₂N(D¹⁸)CO—, —CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D¹⁸)-, —SO₂N(D¹⁸)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, and ND¹⁸;    -   n is an integer from 1 to 10;    -   D², D³, D⁴ and D¹⁸ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D¹⁰ is selected from the group consisting of hydrogen, CF₃,        halogen, CH₃, and OMe;    -   D¹¹, D¹², D¹³, and D¹⁴ are independently selected from the group        consisting of hydrogen, —COD¹⁵D¹⁶, —COND¹⁵D¹⁶, —COOD¹⁷,        —ND¹⁷COND¹⁵; and    -   D¹⁵, D¹⁶, and D¹⁷ are independently selected from the group        consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,        aryl, and heterocycle.

In yet another embodiment, a compound of the invention comprises Formula(XXII), wherein

-   -   X is OH;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of sulfur, CD³, ND⁴,        —N(D¹⁸)CO—, and —CH₂N(D¹⁸);    -   D³, D⁴ and D¹⁸ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D¹⁰ is selected from the group consisting of hydrogen, CF₃,        halogen, CH₃, and OMe;    -   D¹¹ and D¹³ are hydrogen; and    -   D¹² and D¹⁴ are independently selected from the group consisting        of hydrogen, —CONHCH₃, —COOCH₃, and —NHCONH₂.

In still yet another embodiment, a compound of the invention comprisesFormula (XXII), wherein

-   -   X is OH;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of CD³ and —CH₂N(D¹⁸);    -   D³, D⁴ and D¹⁸ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D¹⁰ is selected from the group consisting of hydrogen, CF₃,        halogen, CH₃, and OMe;    -   D¹¹ and D¹³ are hydrogen; and    -   D¹² and D¹⁴ are independently selected from the group consisting        of hydrogen, —CONHCH₃, —COOCH₃, and —NHCONH₂.

Yet another aspect of the invention is a compound of the Formula(XXIII):

wherein

-   -   X is selected from the group consisting of hydrogen, OD², SD²,        ND^(z);    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CD³,        ND⁴, —N(D⁵)CO—, —CH₂N(D⁵)-, —CH₂N(D⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D⁵)-, —SO₂N(D⁵)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, ND⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   D², D³, D⁴, D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CD⁶ and        N;    -   D⁶ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁰ and D²² are selected from the group consisting of hydrogen        and —COOD¹⁵;    -   D²¹ is selected from the group consisting of hydrogen, a five        membered cycloalkyl or heterocyclo ring, and a halogen;    -   D²³ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D¹⁵ is selected from the group consisting of hydrogen,        hydrocarbyl, substituted hydrocarbyl, aryl, and heterocycle;    -   D^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COD^(x),        —COND^(x)D^(x)SO₂D^(x), and —CO₂D^(x); and    -   D^(x) is independently selected from the group consisting of        hydrogen, —ND⁴D⁵, and an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl.

In one embodiment, a compound of the invention comprises Formula(XXIII), wherein

-   -   X is OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, CD³,        —CH₂N(D⁵)CO—; D², D³, D⁴, D⁵ are independently selected from the        group consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   A is CD⁶;    -   D⁶ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁰ and D²² are selected from the group consisting of hydrogen        and —COOD¹⁵;    -   D²¹ is selected from the group consisting of hydrogen, halogen,        and a five membered cycloalkyl or heterocyclo ring;    -   D²³ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl; and    -   D¹⁵ is selected from the group consisting of hydrogen,        hydrocarbyl, substituted hydrocarbyl, aryl, and heterocycle.

In another embodiment, a compound of the invention comprises Formula(XXIII), wherein

-   -   X is OH;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, CD³,        —CH₂N(D⁵)CO—;    -   D³, D⁴, and D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   A is CD⁶;    -   D⁶ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁰ and D²² are selected from the group consisting of hydrogen        and —COOD¹⁵;    -   D²¹ is selected from the group consisting of hydrogen, halogen,        a five membered cycloalkyl or heterocyclo ring;    -   D²³ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl; and    -   D¹⁵ is selected from the group consisting of hydrogen,        hydrocarbyl, substituted hydrocarbyl, aryl, and heterocycle.

In yet another embodiment, a compound of the invention comprises Formula(XXIII), wherein

-   -   X is OH;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, CD³,        —CH₂N(D⁵)CO—;    -   D³, D⁴, and D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   A is CD⁶;    -   D⁶ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁰ and D²² are —COOD¹⁵;    -   D²¹ and D²³ and hydrogen; and    -   D¹⁵ is selected from the group consisting of hydrogen,        hydrocarbyl, substituted hydrocarbyl, aryl, and heterocycle.

In still yet another embodiment, a compound of the invention comprisesFormula (XXIII), wherein

-   -   X is OH;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, CD³,        —CH₂N(D⁵)CO—;    -   D³, D⁴, D⁵, D⁸, and D⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   A is CD⁶;    -   D⁶ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl; D²⁰ and D²² are        hydrogen;    -   D²¹ is selected from the group consisting of hydrogen, halogen,

-   -   wherein G is selected from the group consisting of S, O, CD⁸,        and ND⁹; and    -   D²³ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl.

Still yet another aspect of the invention encompasses a compound ofFormula (XXIV):

wherein

-   -   X is selected from the group consisting of hydrogen, OD², SD²,        and ND^(z);    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CD³,        ND⁴, —N(D⁵)CO—, —CH₂N(D⁵)-, —CH₂N(D⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D⁵)-, —SO₂N(D⁵)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, ND⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   D², D³, D⁴, D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CD⁶ and        N;    -   G is selected from the group consisting of S, O, CD⁸, and ND⁹;    -   D⁶, D⁸ and D⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D²⁴ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁵ and D²⁶ is selected from the group consisting of hydrogen,        —NHCONH₂, —COOMe, and —CONHMe, and D²⁵ and D²⁶ can optionally        form a cycloalkyl or heterocyclo ring;    -   D^(z) is independently selected from the group consisting of        hydrogen hydrocarbyl, substituted hydrocarbyl, —COD^(x),        —COND^(x)D^(x)SO₂D^(x), and —CO₂D^(x); and    -   D^(x) is independently selected from the group consisting of        hydrogen, —ND⁴D⁵, or an optionally substituted alkyl,        cycloalkyl, heterocycle, or aryl.

In one embodiment, a compound of the invention encompasses Formula(XXIV) wherein:

-   -   X is selected from the group consisting of hydrogen, OD², and        SD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CD³,        ND⁴, —N(D⁵)CO—, —CH₂N(D⁵)-, —CH₂N(D⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D⁵)-, —SO₂N(D⁵)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, ND⁵, and an optionally substituted        alkyl, alkene, alkyne, or heterocycle;    -   D², D³, D⁴, D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CD⁶ and        N;    -   G is selected from the group consisting of S, O, CD⁸, and ND⁹;    -   D⁶, D⁸ and D⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D²⁴ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁵ and D²⁶ is selected from the group consisting of hydrogen,        —NHCONH₂, —COOMe, and —CONHMe, or D²⁵ and D²⁶ can optionally        form a cycloalkyl or heterocyclo ring.

In another embodiment, a compound of the invention encompasses Formula(XXIV) wherein:

-   -   X is OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, sulfur, CD³,        ND⁴, —N(D⁵)CO—, —CH₂N(D⁵)-, —CH₂N(D⁵)CO—, CO₂, SO₂, —CH₂O—,        —CH₂S—, CO, —CON(D⁵)-, —SO₂N(D⁵)-, —O(CH₂)_(n)—, —S(CH₂)_(n)—,        —N(CH₂)_(n)—, —(CH₂)_(n)—, and ND⁵;    -   D², D³, D⁴, D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   n is an integer from 1 to 10;    -   A is independently selected from the group consisting of CD⁶ and        N;    -   G is selected from the group consisting of S, O, CD⁸, and ND⁹;    -   D⁶, D⁸ and D⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D²⁴ is selected from the group consisting of hydrogen, halogen,        hydrocarbyl, and substituted hydrocarbyl;    -   D²⁵ and D²⁶ are selected from the group consisting of hydrogen,        —NHCONH₂, —COOMe, and —CONHMe, or D²⁵ and D²⁶ can optionally        form the structure:

In yet another embodiment, a compound of the invention encompasses acompound of Formula (XXIV) wherein:

-   -   X is OD²;    -   Z is selected from the group consisting of O, S, CD³ and ND⁴;    -   Y is selected from the group consisting of oxygen, CD³, ND⁴, and        —CH₂N(D⁵)-;    -   D², D³, D⁴, D⁵ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   A is independently selected from the group consisting of CD⁶ and        N;    -   G is selected from the group consisting of S, O, CD⁸, and ND⁹;    -   D⁶, D⁸ and D⁹ are independently selected from the group        consisting of hydrogen, hydrocarbyl, and substituted        hydrocarbyl;    -   D²⁴ is selected from the group consisting of hydrogen, halogen,        CH₃, CF₃, and OMe;    -   D²⁵ and D²⁶ are selected from the group consisting of hydrogen,        —NHCONH₂, —COOMe, and —CONHMe, or D²⁵ and D²⁶ can optionally        form the structure

In an exemplary alternative of each of the foregoing embodiments, acompound comprising Formula (XXI) is a compound comprising any of theFormulas in Table 21 or 22.

In a further exemplary alternative of each of the foregoing embodiments,a compound of the invention is 4ZFH284 or 4ZFH269 from Table 22.

In certain embodiments, the sugar residue of the above compounds mayencompass any stereoisomer of mannose. In other embodiments, the sugarresidue of the above compounds may encompass any stereoisomer of mannoseother than glucose. In an exemplary embodiment, the sugar residue of theabove compounds is alpha D mannose.

Exemplary methods of synthesizing a compound of the invention aredetailed in the Examples.

A compound of the invention may also be an intermediate in the synthesisof a compound of formula (I)-(XIX). For instance, in one embodiment, acompound of the invention may be an ester intermediate in the synthesisof a compound of formula (I)-(XIX). In another embodiment, a compound ofthe invention may be a boronate ester of a mannoside or a boronic acidester of a mannoside. In yet another embodiment, a compound of theinvention may have the formula (XX), wherein R′ is selected from H,alkyl, or both R′ groups may together form a ring (for instance, see thesynthesis of compound 77 detailed in the Examples below). In stillanother embodiment, a compound of the invention may be a compoundillustrated in Schemes 1, 2, 3, 1^(a), 2^(a), 3^(a), 4^(a), or 5^(a) inthe Examples below.

A compound of the invention may also comprise an imaging agent, such asa fluorescent moiety. For example, see compounds 98 and 99 of Table 15.In an exemplary embodiment, the imaging agent is bound to the sugarportion of a compound of the invention, either directly, or via alinker.

Compounds of the invention may block the function of FimH of the type Ipili of pathogenic bacteria and prevent bacterial adherence and invasionand thus prevent bacterial amplification in the IBC and subsequentspreading and repeated rounds of amplification via new generation IBCs.

FimH functional assays used to measure activity of the compounds areknown to individuals skilled in the art. Non-limiting examples offunctional assays include hemmagglutination titer using guinea pig redblood cells, affinity of binding to FimH, and the ability of thecompounds to prevent biofilm formation.

In some embodiments, activity of the compound is measured usinghemmagglutination titer of guinea pig red blood cells. Hemagglutinationof guinea pig red blood cells by typel piliated UPEC is dependent uponFimH mannose binding ability and serial dilutions allow a quantitativeanalysis. Hemagglutination titer may generally be defined as the amountof compound required for decreasing hemagglutination by 75%. In someembodiments, the hemmagglutination titer of the compound of theinvention may be less than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 μM. In a preferred alternative of the embodiments, thehemmagglutination titer of the compound of the invention may be lessthan about 9 μM. In another preferred alternative of the embodiments,the hemmagglutiantion titer of the compound of the invention may be lessthan about 7 μM. In yet another preferred alternative of theembodiments, the hemmagglutination titer of the compound of theinvention may less than about 1 μM.

In yet other embodiments, activity of the compound may be measured usingthe ability of the compound to prevent or disrupt biofilm formation. Ingeneral, titration curves measuring the ability of a compound inhibitbiofilm formation may be performed to determine the IC₅₀. In someembodiments, the IC₅₀ of the compound may be less than about 700, 600,500, 400, 300, 200 or 100 μM. In other embodiments, the IC₅₀ of thecompound may be less than about 500, 400, 300, 200, 100, 50, 40, 30, 2010, 9, 8, 7, 6, or 5 μM. In preferred embodiments, the IC₅₀ of thecompound may be less than about 20 μM. In other preferred embodiments,the IC₅₀ of the compound may be less than about 9 μM.

II. Combinations

Another aspect of the present invention encompasses a combination of acompound of the invention (described in Section I above) with one ormore bactericidal compounds. In some embodiments, a compound of theinvention may comprise a combination with 1, 2, 3, 4, or 5 bactericidalcompounds. In one embodiment, the bactericidal compound is anantibiotic. Suitable antibiotics are known in the art, and may includeAmikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin,Paromomycin, Geldanamycin, Herbimycin, Carbacephem, Loracarbef,Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil,Cefazolin, Cefalotin, Cefalexin, Cephalosporins, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin,Vancomycin, Telavancin, Clindamycin, Lincomycin, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Troleandomycin, Telithromycin, Spectinomycin, Aztreonam, Furazolidone,Nitrofurantoin, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Temocillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B,Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin,Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin,Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide,Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfamethizole, Sulfamethoxazole (SMZ), Sulfanilimide,Sulfasalazine, Sulfisoxazole, Trimethoprim (TMP),TrimethoprimSulfamethoxazole (such as Bactrim, Septra), Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine,Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin,Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Linezolid,Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin,Rifaximin, Thiamphenicol, or Tinidazole. In an exemplary embodiment, theantibiotic is TMP, SMZ, or a combination thereof.

III. Pharmaceutical Compositions

Yet another aspect of the invention encompasses a pharmaceuticalcomposition. A compound of the invention described in section I abovemay exist in tautomeric, geometric or stereoisomeric forms. The presentinvention contemplates all such compounds, including cis- andtrans-geometric isomers, E- and Z-geometric isomers, R- andS-enantiomers, diastereomers, d-isomers, l-isomers, the racemic mixturesthereof and other mixtures thereof. Pharmaceutically acceptable salts ofsuch tautomeric, geometric or stereoisomeric forms are also includedwithin the invention. The terms “cis” and “trans”, as used herein,denote a form of geometric isomerism in which two carbon atoms connectedby a double bond will each have a hydrogen atom on the same side of thedouble bond (“cis”) or on opposite sides of the double bond (“trans”).Some of the compounds described contain alkenyl groups, and are meant toinclude both cis and trans or “E” and “Z” geometric forms. Furthermore,some of the compounds described contain one or more stereocenters andare meant to include R, S, and mixtures of R and S forms for eachstereocenter present.

In a further embodiment, the inhibitors of the present invention may bein the form of free bases or pharmaceutically acceptable acid additionsalts thereof. The term “pharmaceutically-acceptable salts” are saltscommonly used to form alkali metal salts and to form addition salts offree acids or free bases. The nature of the salt may vary, provided thatit is pharmaceutically acceptable. Suitable pharmaceutically acceptableacid addition salts of compounds for use in the present methods may beprepared from an inorganic acid or from an organic acid. Examples ofsuch inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric,carbonic, sulfuric and phosphoric acid. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which are formic, acetic, propionic, succinic, glycolic, gluconic,lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric,pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, stearic,algenic, algenic, hydroxybutyric, salicylic, galactaric and galacturonicacid. Suitable pharmaceutically-acceptable base addition salts ofcompounds of use in the present methods include metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium and zinc ororganic salts made from N, N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, ethylenediamine, meglumine-(N-methylglucamine)and procaine. All of these salts may be prepared by conventional meansfrom the corresponding compound by reacting, for example, theappropriate acid or base with any of the compounds of the invention.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent.Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed, including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are useful in the preparation of injectables.Dimethyl acetamide, surfactants including ionic and non-ionicdetergents, and polyethylene glycols can be used. Mixtures of solventsand wetting agents such as those discussed above are also useful.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, thecompound is ordinarily combined with one or more adjuvants appropriateto the indicated route of administration. If administered per os, thecompound can be admixed with lactose, sucrose, starch powder, celluloseesters of alkanoic acids, cellulose alkyl esters, talc, stearic acid,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets cancontain a controlled-release formulation as can be provided in adispersion of active compound in hydroxypropylmethyl cellulose. In thecase of capsules, tablets, and pills, the dosage forms can also comprisebuffering agents such as sodium citrate, or magnesium or calciumcarbonate or bicarbonate. Tablets and pills can additionally be preparedwith enteric coatings.

For therapeutic purposes, formulations for parenteral administration maybe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. These solutions and suspensions may beprepared from sterile powders or granules having one or more of thecarriers or diluents mentioned for use in the formulations for oraladministration. The compounds may be dissolved in water, polyethyleneglycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil,sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.Other adjuvants and modes of administration are well and widely known inthe pharmaceutical art. For instance, a compound of the invention may beadministered with a carrier. Non-limiting examples of such a carrierinclude protein carriers and lipid carriers.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The amount of the compound of the invention that may be combined withthe carrier materials to produce a single dosage of the composition willvary depending upon the subject and the particular mode ofadministration. Those skilled in the art will appreciate that dosagesmay also be determined with guidance from Goodman & Goldman's ThePharmacological Basis of Therapeutics, Ninth Edition (1996), AppendixII, pp. 1707-1711 and from Goodman & Goldman's The Pharmacological Basisof Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.

A compound of the invention may also be formulated as a prodrug. Such aprodrug formulation may increase the bioavailability of a compound ofthe invention. In one embodiment, the sugar portion of a compound of theinvention may encompass a prodrug. In another embodiment R¹ may comprisea prodrug. Non-limiting examples of a compound of the inventionformulated as a prodrug include the compounds below:

IV. Methods of the Invention

Compounds of the invention may be used in methods of treating abacterial infection and methods of reducing resistance to a bactericidalcompound in a bacterium.

(a) Methods of Treating a Bacterial Infection

One embodiment of the invention encompasses a method for treatingbacterial infections. As used herein, “treating” refers to preventinginfection in a subject not currently infected, and reducing oreliminating infection in a subject that is currently infected.Generally, such a method comprises administering a pharmaceuticalcomposition comprising a compound of the invention to a subject. As usedherein, “subject” includes any mammal prone to urinary tract infectionsby E. coli. In one embodiment, a subject is prone to recurring UTIs. Insome embodiments, a subject may not have clinical symptoms of a UTI. Insuch embodiments, the subject may have a latent infection. In otherembodiments, a subject may have clinical symptoms of a UTI.

In some embodiments, a compound of the invention may be administered toa subject in combination with a bactericidal compound as described inSection II above. When administered in a combination, a compound of theinvention may be administered before, simultaneously, or afteradministration of a bactericidal compound. When administered before orafter a bactericidal compound, the time between administration of acompound of the invention and a bactericidal compound may be about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, or 60 min. In another embodiment, the time between administration ofa compound of the invention and a bactericidal compound may be about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours.

A compound or pharmaceutical composition of the invention may beadministered by several different means that will deliver atherapeutically effective dose. Such compositions may be administeredorally, parenterally, by inhalation spray, rectally, intradermally,intracisternally, intraperitoneally, transdermally, bucally, as an oralor nasal spray, topically (i.e. powders, ointments or drops), or via aurinary cathetar in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehiclesas desired. Topical administration may also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. The term parenteral as used herein includes subcutaneous,intravenous, intramuscular, or intrasternal injection, or infusiontechniques. In an exemplary embodiment, the pharmaceutical compositionwill be administered in an oral dosage form. Formulation of drugs isdiscussed in, for example, Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H. A.and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, NewYork, N.Y. (1980).

The amount of a compound of the invention that constitutes an “effectiveamount” can and will vary. The amount will depend upon a variety offactors, including whether the administration is in single or multipledoses, and individual subject parameters including age, physicalcondition, size, and weight. Those skilled in the art will appreciatethat dosages may also be determined with guidance from Goodman &Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition(1996), Appendix II, pp. 1707-1711 and from Goodman & Goldman's ThePharmacological Basis of Therapeutics, Tenth Edition (2001), AppendixII, pp. 475-493.

In order to selectively control the release of an inhibitor to aparticular region of the gastrointestinal tract for release, thepharmaceutical compositions of the invention may be manufactured intoone or several dosage forms for the controlled, sustained or timedrelease of one or more of the ingredients. In this context, typicallyone or more of the ingredients forming the pharmaceutical composition ismicroencapsulated or dry coated prior to being formulated into one ofthe above forms. By varying the amount and type of coating and itsthickness, the timing and location of release of a given ingredient orseveral ingredients (in either the same dosage form, such as amulti-layered capsule, or different dosage forms) may be varied.

In an exemplary embodiment, the coating may be an enteric coating. Theenteric coating generally will provide for controlled release of theingredient, such that drug release can be accomplished at some generallypredictable location in the lower intestinal tract below the point atwhich drug release would occur without the enteric coating. In certainembodiments, multiple enteric coatings may be utilized. Multiple entericcoatings, in certain embodiments, may be selected to release theingredient or combination of ingredients at various regions in the lowergastrointestinal tract and at various times.

As will be appreciated by a skilled artisan, the encapsulation orcoating method can and will vary depending upon the ingredients used toform the pharmaceutical composition and coating, and the desiredphysical characteristics of the microcapsules themselves. Additionally,more than one encapsulation method may be employed so as to create amulti-layered microcapsule, or the same encapsulation method may beemployed sequentially so as to create a multi-layered microcapsule.Suitable methods of microencapsulation may include spray drying,spinning disk encapsulation (also known as rotational suspensionseparation encapsulation), supercritical fluid encapsulation, airsuspension microencapsulation, fluidized bed encapsulation, spraycooling/chilling (including matrix encapsulation), extrusionencapsulation, centrifugal extrusion, coacervation, alginate beads,liposome encapsulation, inclusion encapsulation, colloidosomeencapsulation, sol-gel microencapsulation, and other methods ofmicroencapsulation known in the art. Detailed information concerningmaterials, equipment and processes for preparing coated dosage forms maybe found in Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al.(New York: Marcel Dekker, Inc., 1989), and in Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. (Media,Pa.: Williams & Wilkins, 1995).

A bacterium may be contacted with a compound of the invention in vivo,in vitro, in situ, or ex vivo. In some embodiments, a bacterium may bedirectly contacted with the compound of the invention. In otherembodiments, an intracellular bacterium may be contacted with a compoundof the invention. Suitable cells comprising one or more bacteria may begrown, sub-cultured, stored and manipulated using standard techniquesknown to individuals skilled in the art. Cell culture andmicrobiological techniques for growing, culturing, storing, andmanipulating cells comprising one or more bacteria are commonly known inthe art.

(b) Methods of Reducing Bactericidal Resistance

Another method of the invention comprises reducing the resistance of abacterium to a bactericidal compound. Such a method comprises contactinga bacterium resistant to a bactericidal compound with a compound of theinvention. For instance, a subject infected with a bacterium resistantto a bactericidal compound may be administered a compound of theinvention, as described in section IV(a) above. In an exemplaryembodiment, a method comprises contacting a bacterium resistant to anantibiotic with a compound of the invention. In a further exemplaryembodiment, a method comprises contacting a bacterium resistant to TMPor SMZ with a compound of the invention.

Methods of measuring resistance of a bacterium to an antibiotic areknown in the art. For more details, see the examples.

(c) Methods of Treating Catheter-Associated Urinary Tract Infections

In a further embodiment, a method of the invention encompasses a methodfor treating catheter-associated urinary tract infections. As usedherein, “treating” refers to preventing infection in a subject notcurrently infected, and reducing or eliminating infection in a subjectthat is currently infected. Generally, such a method comprisesadministering a pharmaceutical composition comprising a compound of theinvention to a subject. For this embodiment, “subject” refers to anymammal with an indwelling urinary catheter. In one embodiment, a subjectwith a urinary catheter is prone to recurring UTIs. In some embodiments,a subject with a urinary catheter may not have clinical symptoms of aUTI. In such embodiments, the subject may have a latent infection. Inother embodiments, a subject with a urinary catheter may have clinicalsymptoms of a UTI.

In some embodiments, a compound of the invention may be administered toa subject in combination with a bactericidal compound as described inSection II and IV(a) above.

V. Coatings

An additional aspect of the present invention encompasses coatingscomprising a compound of the invention. Such a coating may be used on amedical device to prevent bacterial adherence or infection of the host.Suitable means of coating medical devices are known in the art. In oneembodiment, a catheter may be coated with a compound of the invention.In another embodiment, a urinary catheter may be coated with a compoundof the invention.

VI. Nutritional Supplement

An alternative aspect of the present invention encompasses a nutritionalsupplement that comprises a compound of the invention. Such a supplementmay be used to treat a bacterial infection as described in section IVabove.

Definitions

The term “acyl,” as used herein alone or as part of another group,denotes the moiety formed by removal of the hydroxyl group from thegroup —COOH of an organic carboxylic acid, e.g., RC(O)—, wherein R isR′, R₁O—, R′R₂ N—, or R₁S—, R₁ is hydrocarbyl, heterosubstitutedhydrocarbyl, or heterocyclo and R₂ is hydrogen, hydrocarbyl orsubstituted hydrocarbyl.

The term “acyloxy,” as used herein alone or as part of another group,denotes an acyl group as described above bonded through an oxygenlinkage (—O—), e.g., RC(O)O— wherein R is as defined in connection withthe term “acyl.”

Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to eight carbon atoms in theprincipal chain and up to 20 carbon atoms. They may be straight orbranched chain or cyclic and include methyl, ethyl, propyl, isopropyl,butyl, hexyl and the like.

Unless otherwise indicated, the alkenyl groups described herein arepreferably lower alkenyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain or cyclic and include ethenyl, propenyl, isopropenyl,butenyl, isobutenyl, hexenyl, and the like.

Unless otherwise indicated, the alkynyl groups described herein arepreferably lower alkynyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain and include ethynyl, propynyl, butynyl, isobutynyl,hexynyl, and the like.

The terms “aryl” or “ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Phenyl andsubstituted phenyl are the more preferred aryl.

As used herein, the term “functional group” includes a group of atomswithin a molecule that is responsible for certain properties of themolecule and/or reactions in which it takes part. Non-limiting examplesof functional groups include, alkyl, carboxyl, hydroxyl, amino,sulfonate, phosphate, phosphonate, thiol, alkyne, azide, halogen, andthe like.

The terms “halogen” or “halo” as used herein alone or as part of anothergroup refer to chlorine, bromine, fluorine, and iodine.

The terms “heterocyclo” or “heterocyclic” as used herein alone or aspart of another group denote optionally substituted, fully saturated orunsaturated, monocyclic or bicyclic, aromatic or nonaromatic groupshaving at least one heteroatom in at least one ring, and preferably 5 or6 atoms in each ring. The heterocyclo group preferably has 1 or 2 oxygenatoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring,and may be bonded to the remainder of the molecule through a carbon orheteroatom. Exemplary heterocyclo include heteroaromatics such as furyl,thienyl, pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, orisoquinolinyl and the like. Exemplary substituents include one or moreof the following groups: hydrocarbyl, substituted hydrocarbyl, keto,hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy,aryloxy, halogen, amido, amino, nitro, cyano, thiol, ketals, acetals,esters and ethers.

The term “heteroaromatic” as used herein alone or as part of anothergroup denote optionally substituted aromatic groups having at least oneheteroatom in at least one ring, and preferably 5 or 6 atoms in eachring. The heteroaromatic group preferably has 1 or 2 oxygen atoms, 1 or2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may bebonded to the remainder of the molecule through a carbon or heteroatom.Exemplary heteroaromatics include furyl, thienyl, pyridyl, oxazolyl,pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like. Exemplarysubstituents include one or more of the following groups: hydrocarbyl,substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl,acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino,nitro, cyano, thiol, ketals, acetals, esters and ethers.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms.

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted with ahetero atom such as nitrogen, oxygen, silicon, phosphorous, boron,sulfur, or a halogen atom. These substituents include halogen,carbocycle, aryl, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,hydroxy, protected hydroxy, keto, acyl, acyloxy, nitro, amino, amido,nitro, cyano, thiol, ketals, acetals, esters and ethers.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. Those of skill in the art should, however, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1. Uropathogenic E. coli (UPEC) Pathogenesis in the UrinaryTract

Clinically, it has been presumed that UPEC infection consists of arelatively simple extracellular colonization of the luminal surfaceafter inoculation of fecal flora into the bladder via the urethra. Incontrast, using a murine model of UPEC infection of the UT, theinventors have detailed an unexpectedly complex UPEC pathogenesis cyclethat involves both intracellular and extracellular niches (FIG. 1).Using genetic, biochemical and cell biological approaches together witha variety of imaging techniques including transmission, quickfreeze-deep etch and scanning electron microscopy, as well as confocaland time lapse video microscopy, the inventors discovered that UPECinvade bladder facet cells via a FimH-dependent mechanism (see below).After invasion, cytoplasmic intracellular bacterial communities (IBCs)are formed. Rapid replication of the initial invading bacteria resultsin the formation of an early IBC of loosely-packed rod-shaped bacteria.The bacteria continue to replicate and progress to form a large denselypacked mid-stage IBC of morphologically coccoid bacteria, withbiofilm-like characteristics including positive periodic acid-Schiff(PAS) staining and differential gene expression throughout thecommunity. After the IBC matures, bacteria detach from the biomass,often become filamentous, and spread to neighboring cells forming newgeneration IBCs. Thus, the IBC pathway facilitates massive expansion ofthe invading bacteria in a niche protected from host defenses.Translational studies have shown that the majority of UPEC isolates formIBCs when introduced into the murine bladder and that IBCs andfilamentous bacteria occur in the urine of human UTI patients.Population dynamic studies conducted by the inventors using ex vivogentamicin protection assays demonstrated that ˜10⁴ UPEC of an initial10⁷ inoculum invaded the bladder tissue within 15 minutes afterinfection and that one percent of the invaded bacteria went on to formIBCs, resulting in an average of 100 IBCs per infected mouse bladder. Ifthis is extrapolated to the human situation, innate defenses in thebladder most likely prevent the majority of bacterial inoculation eventsinto the bladder from leading to disease. However, the ramifications ofthe IBC cascade are striking. Invasion of a single infecting bacteriumcan lead to rapid expansion of the infection via IBC formation,replicating within hours to 10⁴ bacteria and even higher numbersfollowed by dispersal of the bacteria from the biomass and spreading toneighboring cells to reinitiate the IBC cascade. This process allows thebacteria to gain a critical foothold. Bacterial descendents of the acuteIBC cascade have been shown using a murine model, to be able to form aquiescent intracellular reservoir (QIR) that can persist, protected fromantibiotics and seemingly undetected by the host immune system evenafter the acute infection is resolved and bacteria are no longerdetectable in the urine. Bacteria in the QIR can later seed a recurrentinfection, manifested by IBC formation, bacteruria and inflammation.

Example 2. FimH as a Therapeutic Target

There are several key implications from understanding UPEC pathogenesis.Mannosides and pilicides that block FimH function will prevent bacterialadherence and invasion and thus prevent bacterial amplification in theIBC and subsequent spreading and repeated rounds of amplification vianew generation IBCs. These compounds will have potent therapeuticactivity by preventing bacterial expansion which may also have theconsequence of eliminating or significantly reducing the QIR thusreducing predisposition to recurrent infection.

Type 1 pili/FimH are critical for UPEC Pathogenesis in the UT.

Type 1 pili are essential cystitis virulence determinants. Usingscanning and high-resolution EM and the mouse cystitis model developedby the inventors, it was shown that adhesive type 1 piliated bacteriaare able to bind and invade host superficial umbrella cells, while UPEClacking type 1 pili are not. Colonization and invasion of the bladderepithelium is dependent on the FimH adhesion located at the distal endof the pilus that binds mannose residues on bladder epithelial cells.High-resolution freeze-dry/deep-etch EM revealed that FimH interactsdirectly with receptors on the luminal surface of the bladder (FIG. 1).Standard gentamicin protection assays of infected tissue culture cellsand ex vivo gentamicin treatment of infected bladders demonstrated thatfimH⁺ type 1 piliated clinical cystitis isolates, but not fimH⁻ mutants,could invade bladder epithelial cells. Using immunohistochemistry andPfim-gfp transcriptional fusions it was demonstrated that type 1 piliare expressed within IBCs. Using high-resolution EM, pilus-like fibersradiating from bacteria and interacting with matrix material within theintracellular IBC were also visualized (FIG. 1). These results combinedwith work showing that type 1 pili are required for biofilm formation inin vitro systems led to the hypothesis that type 1 pili promote IBCformation and/or maintenance. Therefore, an anhydrotetracycline (AHT)inducible fim strain was constructed which can be “pre-piliate” UTI89 invitro by growth in AHT before infecting mouse bladders, allowing theinitial invasion event to normally. However, once inoculated into themouse, AHT is no longer present, fim transcription ceases and piliationis diluted upon each bacterial division. Using this system, the earliestevents of colonization and invasion were identical between the wild typeand conditional strain. However, the inability of the conditional strainto produce type 1 pili intracellularly abolished its ability to formIBCs, as shown by confocal microscopy, and thus dramatically attenuatedvirulence as determined by CFUs at later time points. These resultsstrongly suggest that type 1 pili are required for the survival andproliferation of UPEC within superficial facet cells. Additionally, thisconditional mutant is significantly impaired in its ability to formQIRs, arguing that the bacteria in QIRs are descendents and thusdependent on the acute IBC cascade.

Structural Studies of FimH and its Ligand.

Adhesive type 1 pili are prototypic structures of a family of adhesivefibers produced by diverse Gram-negative bacteria via thechaperone/usher assembly pathway. Using biochemistry, mutationalstudies, nuclear magnetic resonance, and x-ray crystallography, themolecular basis of pili assembled by the chaperone/usher pathway ingram-negative bacteria, including type 1 pili of UPEC, were delineated(FIG. 2) The three dimensional structure of FimH bound to its mannosereceptor was solved in order to gain a molecular snapshot of a criticalinitial event in UTI pathogenesis (FIG. 3).

FimH is a two domain protein, with a receptor binding domain linked to atypical pilin domain that joins the adhesin to the pilus fiber. Thestructure of the complex of the FimC chaperone bound to FimH (which wasbound to D-mannopyranoside) was determined to 2.8 Å resolution. Themannose binding site of FimH is a deep negatively charged pocket at thetip of its receptor-binding domain. The FimH pocket engages in extensivehydrogen bonding to mannose (FIG. 4), which are abundant in theoligosaccharide moieties of uroplakins that coat the lumenal surface ofthe bladder epithelium. A hydrophobic ridge surrounds the mannosebinding pocket in a manner that may facilitate polar interactions withinthe FimH pocket. Mutational studies revealed that each residue iscritical in mannose binding and pathogenesis, emphasizing why the pocketis invariant among UPEC isolates.

Development of Anti-Adhesives.

The FimH-mannose interaction was further investigated in an effort todevelop potential ligand-based antagonists of UTIs. The chitobiose uniton oligomannose was found to bridge various mannose derivatives to theasparagine in the Asn-X-Ser/Thr motif of FimH resulting in higheraffinity binding. Crystallization of FimH in complex with oligomannose-3revealed the mechanism of this higher affinity binding. The non-reducingMan4 anchors into the mannose-binding pocket while the GlcNAc folds overThr51 allowing specific interactions with a hydrophobic tyrosine gate.Heptyl mannoside mimics the GIcNAc tail of oligomannose-3 and extends itfurther to increase interactions outside the binding pocket resulting inhigh affinity binding (Kd=5 nM). Based on the high affinity of heptylmannose for FimH, the ability of heptyl mannose to reduce bacterialinfection in our mouse model of UTI was tested. First, biofilm formationas a surrogate for IBCs formed in the bladder was evaluated. Heptylmannose at 1 mM inhibited UPEC biofilm formation in vitro, suggestingthat the mannose binding properties of the FimH adhesin is required forbiofilm formation. Thus, UPEC strain UTI89 was incubated with heptylmannose prior to inoculation into the bladders of mice. This resulted ina significant attenuation of virulence at 6 hours post-infection at 5 mMheptyl mannose (FIG. 5). The ability of these compounds to significantlyattenuate virulence establishes mannosides as a potential treatment forUTI. Therefore, more potent mannosides that mimic the natural receptorfor FimH but with increased affinity and avidity in order to ultimatelyblock bacterial colonization, invasion, IBC formation and disease weredeveloped as described below.

Example 3. Genetic Analysis of UPEC Bacterial Isolates

Due to the necessity of FimH, it was tested to see if this gene wasunder positive selection. Through sequencing of more than 200 isolates,the majority of which were UTI isolates, it was discovered that residueson FimH are evolving under positive selection, none of which were in themannose binding pocket. These residues are A48, A83 and V184 in UTI89(FIG. 6). Mutants were constructed containing the other naturallyoccurring amino acids commonly found at the positively selectedpositions. A mutation of A83 to a serine resulted in a defect in bindingin vitro, however only a log drop in bacterial titers were observed invivo. The double mutant containing a valine at position 48 and analanine at position 184 resulted in a severe defect in vivo based onbacterial titers and no defect in the ability to bind mannose in vitro.Mutations in the non-positively selected amino acids had no detectableeffect. This study shows that residues outside the binding pocket arealso important to FimH's function in vivo suggesting there is more toFimH than just its ability to bond to the surface of the bladder throughits mannose binding pocket.

Since FimH has been the only essential virulence factor determined todate, it was investigated if the FimH from UTI89 would result inestablishing a more robust infection if it was put into a clinicalisolate containing an altered FimH. A panel of clinical isolates wasexamined for their ability to form IBCs (FIG. 7). Isolates with a valineat position 48 seemed to perform worse in vivo in regards to IBCformation.

Example 4. Role of FimH in Infection

To assess FimH's role in infection, the native FimH of the clinicalstrain acute 4 (FIG. 7) was replaced with the FimH sequence from UTI89under the control of the native promoter. This was done through lambdared recombinase. The acute4 FimH was first deleted and replaced with achloramphenicol (Cm) cassette. The Cm cassette was then replaced withUTI89 FimH. The isolate was then sequenced to confirm proper sequence.

Acute4 and acute4+UTI89FimH were grown 2×24 hours to obtain type 1expression. C3H/HeN mice were then inoculated with 10⁷ of either strain.For bacterial titers (FIG. 8A), bladders were harvested at 6 and 24hours post-infection. At 6 hours post-infection there was no significantdifference in bacterial load between the two stains and both strainswere much lower that a typical UTI89 6 hour titer (˜10⁶). However, at 24hours post-infection, acute4+UTI89FimH significantly colonized themurine bladder more than actue 4 (p<0.001 by Mann-Whitney). 24 hourtiters in the acute4+UTI89FimH isolate were equivalent to what istypically observed with UTI89. For IBC formation (FIG. 8B), LacZstaining was performed at 6 hours post-infection. No IBCs were observedin either isolate.

Example 5. Synthesis of Compounds

The compounds of the present invention may be prepared in a number ofways well known to one skilled in the art of organic synthesis. Morespecifically, the novel compounds of this invention may be preparedusing the reactions and techniques described herein. In the descriptionof the synthetic methods described below, it is to be understood thatall proposed reaction conditions, including choice of solvent, reactionatmosphere, reaction temperature duration of the experiment and workupprocedures, are chosen to be the conditions standard for that reaction.It is understood by one skilled in the art of organic synthesis that thefunctionality present on various portions of the molecule must becompatible with the reagents and reactions proposed. Such restrictionsto the substituents, which are not compatible with the reactionconditions, will be apparent to one skilled in the art and alternatemethods must then be used. Unless otherwise stated, the startingmaterials for the examples contained herein are either commerciallyavailable or are readily prepared by standard methods from knownmaterials. The compounds of Formula (I) and (II) may be synthesizedthrough standard organic chemistry methodology and purification known tothose trained in the skill and art by using commercially availablestarting materials and reagents. General procedures for synthesizing thecompounds of the invention are as follows:

The chemical synthesis of compounds with the general Formula (I) and(II) is achieved using the routes described in Scheme 1 starting fromcommercially available α-D-mannose, α-D-mannose pentaacetate (Route A),or α-methyl-D mannose (Route B). Shown in Scheme 1A, Lewis-acid mediatedglycosidation of α-D-mannose pentaacetate followed by acetate esterhydrolysis with sodium methoxide yields glycosides of Formula (I).Further chemical transformation of the substituent R¹ may beaccomplished using standard synthetic organic procedures such as, butnot limited to, metal-mediated aryl Suzuki coupling reactions througharyl bromide or aryl boronate ester glycosides, shown in Scheme 2A and2B, respectively, to produce exemplified compounds of Formula (I)described in Tables 1-9. All compounds are also listed in Table 15.

The compounds of Formula (I) may be transformed into compounds ofFormula (II) as described in Scheme 3 by employing various functionalgroups (R¹) and linkers (L), such as, but not limited to, alkyl ethers,esters, amines and amides.

The invention is further described with reference to the followingillustrative examples in which, unless stated otherwise:

-   (i) temperatures are given in degrees Celsius (° C.); operations are    carried out at room temperature or ambient temperature, that is, in    a range of 18-25° C., unless otherwise stated;-   (ii) solutions are dried over anhydrous sodium sulphate or magnesium    sulphate; evaporation of organic solvent is carried out using a    rotary evaporator under reduced pressure (4.5-30 mmHg) with a bath    temperature of up to 60° C.;-   (iii) chromatography means flash chromatography on silica gel; thin    layer chromatography (TLC) is carried out on silica gel plates;-   (iv) in general, the course of reactions are followed by TLC or    liquid chromatography/mass spectroscopy (LC/MS) and reaction times    are given for illustration only;-   (v) final products have satisfactory proton nuclear magnetic    resonance (NMR) spectra and/or mass spectra data;-   (vi) yields are given for illustration only and are not necessarily    those which can be obtained by diligent process development;    preparations are repeated if more material is required;-   (vii) when given, nuclear magnetic resonance (NMR) data is in the    form of delta (o) values for major diagnostic protons, given in part    per million (ppm) relative to tetra methylsilane (TMS) as an    internal standard, determined at 300 MHz ind₃-MeOD unless otherwise    stated;-   (viii) chemical symbols have their usual meanings;-   (ix) solvent ratio is given in volume:volume (v/v) terms;-   (x) Purification of the compounds is carried out using one or more    of the following methods:    -   a) flash chromatography on regular silica gel;    -   b) flash chromatography on silica gel using an MPLC separation        system (Teledyne ISCO):prepacked normal phase flash column        cartridge, flow rate, 10-80 ml/min;    -   c) Preparatory HPLC system using a reverse-phase C18 column,        100×20 mm, 5 μM (or larger) and eluting with combinations of        water (0.1% TFA) and MeCN (0.1% TFA) as the mobile phase;-   (xi) the following abbreviations have been used:    -   Ac acetyl;    -   CIV concentrated in vacuo;    -   RTandrt room temperature;    -   BOC tert-butoxycarbonyl;    -   HATU 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate;    -   DIPEA N,N-diisopropylethylamine;    -   CBZ benzyloxycarbonyl;    -   Bn benzyl;    -   DCM dichloromethane;    -   DMF N,N-dimethylformamide;    -   DMSO dimethylsulfoxide;    -   NMP N-methyl-2-pyrrolidinone;    -   EtOAc ethyl acetate;    -   ether diethyl ether;    -   EtOH ethanol;    -   THF tetrahydrofuran;    -   MeOH methanol;    -   MeCN acetontrile;    -   TFA trifluoracetic acid; and    -   TEA triethylamine.

1. General Procedures for the Preparation of Mannosides when α-D-MannosePentaacetate is Used as the Starting Material: Methyl3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoate(29)

1.1

1.1 Methyl3-[4-[(2R,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoate

Under nitrogen atmosphere, at 0° C. boron trifluoride diethyl etherate(0.128 g, 0.90 mmol) was added dropwise into the solution of α-D-mannosepentaacetate (0.120 g, 0.3 mmol) and methyl 3-(4-hydroxyphenyl)benzoate(0.140 g, 0.6 mmol) in 6 ml of CH₂Cl₂. After a few mins the mixture washeated to reflux and kept stirring for more than 36 hrs. The reactionwas then quenched with water and extracted with CH₂Cl₂. The CH₂Cl₂ layerwas collected, dried with Na₂SO₄, concentrated. The resulting residuewas purified by silica gel chromatography with hexane/ethyl acetatecombinations as eluent, giving rise to methyl 3-[4-[(2R, 3S,4S, 5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoate(0.136 g) in 81% yield. ¹H NMR (300 MHz, CDCl₃) σ ppm 8.23 (m, 1H), 7.99(m, 1H), 7.74 (m, 1H), 7.57 (m, 2H), 7.50 (t, J=7.8 Hz, 1H), 7.18 (m,2H), 5.59 (dd, J=3.6, 9.9 Hz, 1H), 5.58 (d, J=1.5 Hz, 1H), 5.48 (dd,J=2.1, 3.3 Hz, 1H), 5.39 (t, J=10.2 Hz, 1H), 4.30 (dd, J=5.4, 12.3 Hz,1H), 4.06˜4.15 (m, 2H), 3.95 (s, 3H), 2.15 (s, 3H), 2.06 (s, 3H), 2.05(s, 3H), 2.04 (s, 3H); MS (ESI): found: [M+H]⁺, 559.1.

1.2 Methyl3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoate(29)

Methyl 3-[4-[(2 R, 3S,4S, 5 R, 6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoate(0.120 g) was stirred in 6 mL of methanol with catalytic amount ofsodium methoxide (0.02 M) at room temperature overnight. H⁺ exchangeresin (DOWEX 50WX4-100) was added to neutralize the mixture. The resinwas filtered off and the filtrate was concentrated, then dried in vacuogiving rise to pure product 29 (0.084 g) in quantitative yield. ¹H NMR(300 MHz, CD₃OD) δ ppm 8.21 (m, 1H), 7.95 (m, 1H), 7.83 (m, 1H), 7.59(m, 2H), 7.53 (m, 1H), 7.23 (m, 2H), 5.55 (d, J=1.8 Hz, 1H), 4.03 (dd,J=1.8, 3.3 Hz, 1H), 3.91˜3.96 (m, 4H), 3.70˜3.82 (m, 3H), 3.59˜3.65 (m,1H); MS (ESI): found: [M+H]⁺, 391.1. (see Table 1).

2. Procedures for the preparation of mannosides when2,3,4,6-tetra-O-benzyl-1-acetyl-α-D-mannopyranoseis used as the startingmaterial:[3-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]acetate (42)

2.1[3-[(2R,3S,4S,5R,6R)-3,4,5-Tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]oxyphenyl]acetate: Under nitrogen atmosphere, at 0° C. boron trifluoride diethyletherate (0.051 g, 0.36 mmol) was added dropwise into the solution of2,3,4,6-tetra-O-benzyl-1-acetyl-α-D-mannopyranose (0.106 g, 0.18 mmol)and resorcinol monoacetate (0.055 g, 0.36 mmol) in 7 ml of CH₂Cl₂. Themixture was stirred at 0° C. and monitored by TLC. The reaction was thenquenched with water and extracted with CH₂Cl₂. The CH₂Cl₂ layer wascollected, dried with Na₂SO₄, concentrated. The resulting residue waspurified by silica gel chromatography with hexane/ethyl acetatecombinations as eluent to give[3-[(2R,3S,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]oxyphenyl]acetate (0.093 g) in 75% yield. ¹H NMR (300 MHz, CDCl₃) δ ppm 7.15˜7.40(m, 21H), 6.88˜6.91 (m, 1H), 6.81 (t, J=2.4 Hz, 1H), 6.73˜6.76 (m, 1H),5.58 (d, J=1.8 Hz, 1H), 4.90 (d, J=10.8 Hz, 1H), 4.78 (s, 2H), 4.64˜4.69(m, 3H), 4.52 (d, J=10.8 Hz, 1H), 4.45 (d, J=12.3 Hz, 1H), 4.05˜4.18 (m,2H), 3.94 (t, J=2.4 Hz, 1H), 3.77˜3.85 (m, 2H), 3.64˜3.70 (m, 1H), 2.27(s, 3H). MS (ESI): found: [M+Na]⁺, 697.2.

2.2[3-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]acetate (42)

Under hydrogen atmosphere[3-[(2R,3S,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]oxyphenyl] acetate (0.085 g, 0.13 mmol) was stirredwith Pd/C (10 wt. %) (0.132 g, 0.063 mmol) in ethanol (6 mL) and ethylacetate (6 mL). The reaction was monitored by TLC until it went intocompletion. The mixture was filtered through a celite plug. The filtratewas concentrated then dried in vacuo furnishing pure 42 (0.040 g) inquantitative yield. ¹H NMR (300 MHz, CD₃OD) δ ppm 7.30 (t, J=8.1 Hz,1H), 7.00 (m, 1H), 6.90 (t, J=2.1 Hz, 1H), 6.76 (m, 1H), 5.47 (d, J=1.8Hz, 1H), 4.00 (dd, J=1.8, 3.3 Hz, 1H), 3.88 (dd, J=3.3, 9.3 Hz, 1H),3.68˜3.79 (m, 3H), 3.54˜3.61 (m, 1H). MS (ESI): found: [M+H]⁺, 314.9.(see Table 2).

3. Procedure for the Preparation of5-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzene-1,3-dicarboxylicacid (45)

35 (0.025 g, 0.056 mmol) was added into 7 mL methanol. Then 0.20 M NaOHaqueous (3 mL) was added. The mixture was stirred at rt overnight. H⁺exchange resin (DOWEX 50WX4-100) was added to neutralize the mixture.The resin was filtered off and the filtrate was concentrated, then driedin vacuo giving rise to pure product 45 (0.023 g) in quantitative yield.¹H NMR (300 MHz, METHANOL-d₄) δ ppm 3.59-3.67 (m, 1H) 3.70-3.84 (m, 3H)3.94 (dd, J=3.30, 9.30 Hz, 1H) 4.05 (dd, J=3.30, 1.80 Hz, 1H) 5.56 (d,J⁼1.80 Hz, 1H) 7.26 (m, 2H) 7.63 (m, 2H) 8.41 (d, J⁼1.50 Hz, 2H) 8.57(t, J⁼1.50 Hz, 1H). MS (ESI): found: [M+Na]⁺, 443.0. (see Table 3).

4. Procedure for the preparation ofN1,N3-dimethyl-5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzene-1,3-dicarboxamide(50)

35 (0.050 g, 0.11 mmol) was stirred with 15 mL of MeNH₂/EtOH (33 wt. %)at rt for 40 hrs. The solvent was removed and the residue was dried invacuo to afford pure 50 (0.050 g) in quantitative yield. ¹H NMR (300MHz, METHANOL-d₄) δ ppm 2.96 (s, 6H) 3.61-3.66 (m, 1H) 3.67-3.84 (m, 3H)3.86-3.97 (m, 1H) 4.04 (dd, J⁼3.30, 1.92 Hz, 1H) 5.56 (d, J⁼1.92 Hz, 1H)7.21-7.34 (m, 2H) 7.63-7.74 (m, 2H) 8.13-8.26 (m, 3H). MS (ESI): found:[M+H]⁺, 447.4. (see Table 4).

5. Procedures for the Preparation of Biphenyl Mannoside DerivativesThrough Suzuki Coupling Reaction:3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzonitrile(62)

5.1[(2R,3S,4S,5R,6R)-3,4,5-Triacetoxy-6-[4-(3-cyanophenyl)phenoxy]tetrahydropyran-2-yl]methylacetate: Under nitrogen atmosphere, the mixture of acetyl-protected4-bromophenyl α-D-mannoside (0.101 g, 0.2 mmol), m-cyanophenylboronicacid (0.044 g, 0.3 mmol), cesium carbonate (0.196 g, 0.6 mmol) andtetrakis(triphenylphosphine)palladium (0.023 g, 0.02 mmol) indioxane/water (5 mL/1 mL) was heated at 80° C. with stirring for 1 h.The solvent was removed and the resulting residue was purified by silicagel chromatography with hexane/ethyl acetate combinations as eluent togive[(2R,3S,4S,5R,6R)-3,4,5-triacetoxy-6-[4-(3-cyanophenyl)phenoxy]tetrahydropyran-2-yl]methylacetate (0.080 g) in 76% yield. ¹H NMR (300 MHz, CDCl₃) δ ppm 7.82 (t,J=1.5 Hz, 1H), 7.76 (dt, J=7.69, 1.65 Hz, 1H), 7.587.65 (m, 1H),7.477.57 (m, 3H), 7.16˜7.24 (m, 2H), 5.55˜5.65 (m, 2H), 5.47 (dd,J=3.57, 1.92 Hz, 1H), 5.39 (t, J=9.9 Hz, 1H), 4.25˜4.34 (m, 1H),4.04˜4.17 (m, 2H), 2.22 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s,3H). MS (ESI): found: [M+Na]⁺, 548.7.

5.23-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzonitrile(62)

[(2R,3S,4S,5R,6R)-3,4,5-Triacetoxy-6-[4-(3-cyanophenyl)phenoxy]tetrahydropyran-2-yl]methylacetate (0.075 g) was stirred in 6 mL of methanol with catalytic amountof sodium methoxide (0.02 M) at room temperature overnight. H⁺ exchangeresin (DOWEX 50WX4-100) was added to neutralize the mixture. The resinwas filtered off and the filtrate was concentrated, then dried in vacuogiving rise to pure product 62 (0.045 g) in 88% yield. ¹H NMR (300 MHz,CDSOD) δ ppm 3.55˜3.65 (m, 1H), 3.673.83 (m, 3H), 3.84˜3.99 (m, 1H),4.03 (dd, J=3.43, 1.79 Hz, 1H), 5.55 (J=1.65 Hz, 1H), 7.16˜7.33 (m, 2H),7.54˜7.75 (m, 4H), 7.83˜8.01 (m, 2H). MS (ESI): found: [M+H]⁺, 358.3.(see Table 5)

6. Procedures for the Preparation of Biphenyl Mannoside DerivativesThrough Suzuki Coupling Reaction: Methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]pyridine-3-carboxylate (77)

6.1[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]acetate

Under nitrogen atmosphere, the mixture of acetyl-protected 4-bromophenylδ-D-mannoside (2.791 g, 5.55 mmol), bis(pinacolato)diboron (1.690 g,6.66 mmol), potassium acetate (2.177 g, 22.18 mmol) and(1.1′-bis(diphenylphosohino)ferrocene)dichloropalladium(II) (0.244 g,0.33 mmol) in DMSO (50 ml) was heated at 80° C. with stirring for 2.5 h.The solvent was removed and the resulting residue was purified by silicagel chromatography with hexane/ethyl acetate combinations as eluent togive[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]acetate (2.48 g) in 81% yield. ¹H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.33(s, 12H) 1.98-2.12 (m, 9H) 2.20 (s, 3H) 3.93-4.19 (m, 2H) 4.21-4.36 (m,1H) 5.32-5.42 (m, 1H) 5.45 (dd, J⁼3.57, 1.92 Hz, 1H) 5.51-5.62 (m, 2H)7.00-7.15 (m, 2H) 7.67-7.84 (m, 2H). MS (ESI): found: [M+Na]⁺, 573.2.

6.2 Methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]pyridine-3-carboxylate(77)

Under nitrogen atmosphere, the mixture of[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]acetate (0.132 g, 0.24 mmol), methyl 5-bromonicotinate (0.043 g, 0.2mmol), cesium carbonate (0.196 g, 0.6 mmol) andtetrakis(triphenylphosphine)palladium (0.023 g, 0.02 mmol) indioxane/water (5 mL/1 mL) was heated at 80° C. with stirring for 1 h.After cooling down, the mixture was filtered through silica gel columnto remove the metal catalyst and salts with hexane/ethyl acetate (2/1)containing 2% triethylamine as eluent. The filtrate was concentrated,and then dried in vacuo. Into the residue, 6 mL of methanol withcatalytic amount of sodium methoxide (0.02 M) was added and the mixturewas stirred at room temperature overnight. H⁺ exchange resin (DOWEX50WX4-100) was added to neutralize the mixture. The resin was filteredoff and the filtrate was concentrated. The resulting residue waspurified by silica gel chromatography with CH₂Cl₂/MeOH combinationscontaining 2% NH₃/H₂O as eluent, giving rise to 77 (0.031 g) in 40%yield. ¹H NMR (300 MHz, CD₃OD) δ ppm 3.53-3.65 (m, 1H) 3.67-3.83 (m, 3H)3.89-3.96 (m, 1H) 3.99 (s, 3H) 4.04 (dd, J⁼3.43, 1.79 Hz, 1H) 5.57 (d,J⁼1.92 Hz, 1H) 7.22-7.37 (m, 2H) 7.58-7.73 (m, 2H) 8.54 (t, J⁼2.06 Hz,1H) 8.97 (d, J⁼2.20 Hz, 1H) 9.04 (d, J⁼1.92 Hz, 1H). MS (ESI): found:[M+H]⁺, 392.1. (see Table 6).

7. Procedure for the Preparation of Mannosides Via Amide CouplingReaction

3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]-N-[2-[2-[2-[[3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoyl]amino]ethoxy]ethoxy]ethyl]benzamide(82)

Under nitrogen atmosphere, at 0° C. anhydrous DMF (5 mL) was added intothe RB flask containing 44 (0.062 g, 0.165 mmol) and HATU (0.069 g,0.182 mmol). After stirring for 10 min, 1,2-bis(2-aminoethoxy)ethane(0.0123 g, 0.083 mmol), then N,N-diisopropylethylamine (0.024 g, 0.182mmol) were added. The mixture was stirred overnight while being warmedto rt naturally. The solvent was removed and the residue was purified byHPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1% TFA)) togive 82 (0.044 g) in 82% yield. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm3.51-3.65 (m, 6H), 3.65-3.82 (m, 14H), 3.88-3.96 (m, 2H), 4.03 (dd,J=3.57, 1.92 Hz, 2H), 5.52 (d, J=1.65 Hz, 2H), 7.14-7.24 (m, 4H),7.40-7.50 (m, 2H), 7.53-7.63 (m, 4H), 7.66-7.77 (m, 4H), 8.00 (t, J⁼1.65Hz, 2H). MS (ESI): found: [M+H]⁺, 865.9. (see Table 7).

8. Procedures for the Preparation of Fluorescent Mannoside (99 and 100)

8.1[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(9Hfluoren-9-ylmethoxycarbonylamino)butoxy]tetrahydropyran-3-yl]acetate

Under nitrogen atmosphere, at 0° C. boron trifluoride diethyl etherate(0.115 g, 0.81 mmol) was added dropwise into the solution of α-D-mannosepentaacetate (0.107 g, 0.27 mmol) and 4-(Fmoc-amino)-1-butanol (0.168 g,0.54 mmol) in 5 ml of CH₂Cl₂. After a few mins the mixture was heated toreflux and kept stirring for more than 36 hrs. The reaction was thenquenched with water and extracted with CH₂Cl₂. The CH₂Cl₂ layer wascollected, then dried with Na₂SO₄, filtered, and concentrated. Theresulting residue was purified by silica gel chromatography withhexane/ethyl acetate combinations as eluent to give[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(9H-fluoren-9-ylmethoxycarbonylamino)butoxy]tetrahydropyran-3-yl]acetate (0.106 g) in 60% yield. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.48˜1.79(m, 4H), 2.01 (s, 3H), 2.05 (s, 3H), 2.11 (s, 3H), 2.17 (s, 3H), 3.25(m, 2H), 3.50 (m, 1H), 3.73 (m, 1H), 3.91˜4.04 (m, 1H), 4.05˜4.18 (m,1H), 4.18˜4.37 (m, 2H), 4.41 (d, J=6.87 Hz, 2H), 4.74˜4.94 (m, 2H),5.17˜5.41 (m, 3H), 7.29˜7.37 (m, 2H), 7.37˜7.47 (m, 2H), 7.61 (d, J=7.42Hz, 2H), 7.78 (d, J=7.42 Hz, 2H). MS (ESI): found: [M+H]⁺, 642.2.

8.2(2S,3S,4S,5S,6R)-2-(4-aminobutoxy)-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol(98)

[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(9H-fluoren-9-ylmethoxycarbonylamino)butoxy]tetrahydropyran-3-yl]acetate (0.106 g, 0.165 mmol) was stirred in 6 mL of methanol withcatalytic amount of sodium methoxide (0.02M) at rt overnight. Thesolvent was removed and the residue was purified by silica gelchromatography with combination of ammonia aqueous/methanol as eluent toafford 98 (0.036 g) in 88% yield. ¹H NMR (300 MHz, CD₃OD) δ ppm1.46˜1.78 (m, 4H), 2.61˜2.88 (m, 2H), 3.41˜3.55 (m, 2H), 3.59 (t, J=9.3Hz, 1H), 3.65˜3.87 (m, 5H), 4.75 (d, J=1.65 Hz, 1H). MS (ESI): found:[M+H]⁺, 252.1.

8.3 Fluorescent Mannosides (99 and 100)

The mixture of 98 (0.018 g, 0.070 mmol), 5-(and-6)-carboxyfluoresceinsuccinimidyl ester (0.022 g, 0.046 mmol) andtriethylamine (0.058 g, 0.57 mmol) in DMF (2 mL) was stirred overnight.After removing the solvent, the residue was purified by silica gelchromatography with dichloromethane/methanol combination as eluent,giving rise to 99 and 100 (0.023 g) in 82% yield. MS (ESI): found:[M+H]⁺, 610.6. (see Table 8).

9. Procedures for the Preparation of(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-(4-phenyltriazol-1-yl)tetrahydropyran-3,4,5-triol(101)

9.1 [(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-phenyltriazol-1-yl)tetrahydropyran-3-yl]acetate

Under nitrogen atmosphere, EtOH/H₂O (4 mL/1 mL) was added into the RBflask containing α-azido-D-mannose tetraacetate (0.075 g, 0.2 mmol),phenyl acetylene (0.026 g, 0.24 mmol), copper (II) sulfate pentahydrate(0.01 g, 0.04 mmol) and sodium ascorbate (0.016 g, 0.08 mmol). Themixture was stirred at room temperature overnight. The solvent wasremoved and the residue was purified by silica gel chromatography withhexane/ethyl acetate combination as eluent, giving rise to[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-phenyltriazol-1-yl)tetrahydropyran-3-yl]acetate (0.030 g) in 31% yield. ¹H NMR (300 MHz, CHLOROFORM-d) δ ppm2.05-2.12 (m, 9H) 2.20 (s, 3H) 3.89-3.99 (m, 1H) 4.08 (dd, J⁼12.50, 2.61Hz, 1H) 4.40 (dd, J⁼12.50, 5.36 Hz, 1H) 5.40 (t, J=8.79 Hz, 1H)5.95-6.04 (m, 2H) 6.07 (d, J=2.75 Hz, 1H) 7.34-7.53 (m, 3H) 7.79-7.92(m, 2H) 7.96 (s, 1H). MS (ESI): found: [M+Na]+, 498.1.

9.2(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-(4-phenyltriazol-1-yl)tetrahydropyran-3,4,5-triol(101)

[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-phenyltriazol-1-yl)tetrahydropyran-3-yl]acetate (0.028 g, 0.059 mmol) was stirred in 6 mL of methanol withcatalytic amount of sodium methoxide (0.02 M) at room temperatureovernight. H⁺ exchange resin (DOWEX 50WX4-100) was added to neutralizethe mixture. The resin was filtered off and the filtrate wasconcentrated, then dried in vacuo giving rise to pure product 101 (0.015g) in quantitative yield. ¹H NMR (300 MHz, CD₃OD) δ ppm 3.34-3.44 (m,1H) 3.70-3.92 (m, 3H) 4.12 (dd, J⁼8.52, 3.57 Hz, 1H) 4.71-4.80 (m, 1H)6.08 (d, J⁼2.75 Hz, 1H) 7.30-7.41 (m, 1H) 7.41-7.51 (m, 2H) 7.77-7.90(m, 2H) 8.51 (s, 1H). (ESI): found: [M+Na]⁺, 330.2. (see Table 9)

10. Procedures for the Preparation of Methyl3-[4-[[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]amino]phenyl]benzoate(102)

α-D-mannose (0.360 g, 2 mmol) and methyl 4′-aminobiphenyl-3-carboxylate(0.454 g, 2 mmol) in ethanol (5 mL) was heated to 55° C. for 17 h. Aftercooling down to rt, the white precipitate formed was collected byfiltration. The precipitate was washed with ethanol (3 mL) two times,then dried in vacuo to afford pure 102 in 77% yield. ¹H NMR (300 MHz,ACETONITRILE-d3 and D2O) δ ppm 3.28-3.38 (m, 1H) 3.54-3.59 (m, 2H)3.64-3.71 (m, 2H) 3.86 (s, 3H) 3.88-3.92 (m, 1H) 4.89 (d, J=1.10 Hz, 1H)6.80-6.91 (m, 2H) 7.44-7.58 (m, 3H) 7.75-7.90 (m, 2H) 8.11-8.20 (m, 1H).(ESI): found: [M+H]⁺, 390.1.

11. Procedures for the Preparation of methyl3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]sulfanylphenyl]benzoate(103)

11.1[(2R,3S,4S,5R,6R)-4,5-Diacetoxy-6-(acetoxymethyl)-2-(4-bromophenyl)sulfanyl-tetrahydropyran-3-yl]acetate

Under nitrogen atmosphere, at 0° C. boron trifluoride diethyl etherate(0.427 g, 3.0 mmol) was added dropwise into the solution of α-D-mannosepentaacetate (0.390 g, 1.0 mmol) and 4-bromobenzenethiol (0.378 g, 2.0mmol) in 6 ml of CH₂Cl₂. After a few mins the mixture was warmed to rtand kept stirring for 48 hrs. The reaction was then quenched with waterand extracted with CH₂Cl₂. The CH₂Cl₂ layer was collected, dried withNa₂SO₄, filtered, and concentrated. The resulting residue was purifiedby silica gel chromatography with hexane/ethyl acetate combinations aseluent, giving rise to[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromophenyl)sulfanyl-tetrahydropyran-3-yl]acetate (0.40 g) in 77% yield. ¹H NMR (300 MHz, CHLOROFORM-d) δ ppm2.01-2.10 (m, 9H) 2.16 (s, 3H) 4.10 (dd, J=12.09, 2.47 Hz, 1H) 4.30 (dd,J=12.09, 6.04 Hz, 1H) 4.43-4.61 (m, 1H) 5.24-5.40 (m, 2H) 5.43-5.52 (m,2H) 7.32-7.39 (m, 2H) 7.42-7.49 (m, 2H). MS (ESI): found: [2M+H]⁺,1039.1.

11.2 Methyl3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]sulfanylphenyl]benzoate(103)

Under nitrogen atmosphere, the mixture of[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromophenyl)sulfanyl-tetrahydropyran-3-yl]acetate (0.20 g, 0.39 mmol), 3-methoxycarbonylphenylboronic acid (0.106g, 0.59 mmol), cesium carbonate (0.381 g, 1.17 mmol) andtetrakis(triphenylphosphine)palladium (0.045 g, 0.04 mmol) indioxane/water (5 mL/1 mL) was heated at 80° C. with stirring for 1 h.After cooling down, the mixture was filtered through silica gel columnto remove the metal catalyst and salts with hexane/ethyl acetate (2/1)as eluent. The filtrate was concentrated, and then dried in vacuo. Intothe residue, 6 mL of methanol with catalytic amount of sodium methoxide(0.02 M) was added and the mixture was stirred at room temperatureovernight. H⁺ exchange resin (DOWEX 50WX4-100) was added to neutralizethe mixture. The resin was filtered off and the filtrate wasconcentrated. The resulting residue was purified by HPLC (C18, 15*150 mmcolumn; eluent: acetonitrile/water (0.1% TFA)) to give 103 (0.095 g) in63% yield. ¹H NMR (300 MHz, CD³OD) δ ppm 3.66-3.91 (m, 4H) 3.94 (s, 3H)4.01-4.16 (m, 2H) 5.51 (d, J=1.37 Hz, 1H) 7.51-7.69 (m, 5H) 7.81-7.93(m, 1H) 8.00 (dt, J=7.83, 1.44 Hz, 1H) 8.24 (t, J⁼1.65 Hz, 1H). MS(ESI): found: [M nd+ H]⁺, 407.1.

12. Procedures for the Preparation of methyl3-[4-[[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-carbonyl]amino]phenyl]benzoate(104)

The solution of α-cyano-D-mannose tetraacetate (0.107 g, 0.3 mmol) in25% hydrochloric acid was heated at 50° C. for 48 h. The solvent wasremoved. Water (10 ml) and H⁺ exchange resin (DOWEX 50WX4-100) was addedand kept stirring for 5 mins. The resin was filtered off and thefiltrate was concentrated, and then dried in vacuo. Into the resultingresidue, HATU (0.274 g, 0.72 mmol) and anhydrous DMF (6 ml) were addedat 0° C. After stirring for 10 min, methyl4′-aminobiphenyl-3-carboxylate (0.164 g, 0.72 mmol), thenN,N-diisopropylethylamine (0.233 g, 1.80 mmol) were added. The mixturewas stirred overnight while being warmed to rt naturally. The solventwas removed and the residue was purified by silica gel chromatographywith CH₂Cl₂/MeOH combinations as eluent to give 104 (0.066 g) in 52%yield. ¹H NMR (300 MHz, CD₃OD) δ ppm 3.50 (dt, J=4.67, 2.33 Hz, 1H)3.58-3.65 (m, 2H) 3.76 (dd, J⁼11.81, 7.42 Hz, 1H) 3.86-4.03 (m, 4H) 4.52(t, J=2.61 Hz, 1H) 4.57 (d, J=2.75 Hz, 1H) 7.55 (t, J=7.69 Hz, 1H)7.60-7.68 (m, 2H) 7.68-7.76 (m, 2H) 7.82-7.91 (m, 1H) 7.97 (dt, J=7.69,1.37 Hz, 1H) 8.21-8.28 (m, 1H). MS (ESI): found: [M+H]⁺, 418.1.

Example 6. Structure-Based Drug Design and Optimization of MannosideBacterial FimH Antagonists

As shown in Scheme 1, α-D-mannoside derivatives were prepared usingtraditional Lewis acid mediated glycosidation. ³Reaction of acylatedα-D-(+)-mannose 1a with a variety of phenols and BF₃—OEt₂ resulted inexclusive formation of the α-isomer mannosides 2. Subsequent deacylationwith NaOMe in methanol gave the desired aryl mannosides 3-8 in goodyield. Biological activity against FimH was evaluated using a guinea pigred blood cell-based hemagglutination (HA) assay (ref) in which theconcentration of the mannoside on reducing agglutination by 50% was usedas the primary endpoint (50% HA titer). This assay was preferred to asimple FimH binding assay for screening and developing SAR since itassesses the compound's ability to prevent bacterially-mediated adhesiondirectly in a cellular assay.

As shown in Table 10, it was found that the general trend that 2- and3-substitution was optimal for potency relative to 4-substitution inmost examples with the exception of the acyl anilines 3q-s in which thistrend was dramatically reversed. Ortho-substituted chlorophenyl 3g,cyanophenyl 3m, and meta-substituted methyl ester 3j all showed animpressive greater than 5-fold improvement in potency relative to parentphenyl mannoside 3a. Interestingly, carboxylic acid 3l lost 10-foldpotency relative to matched pair methyl ester 3j showing a 50% HA titerof only 60 μM. Incorporation of an additional methyl ester in the5-position of 3j, as with compound 4, resulted in a relatively large3-fold enhancement in potency. Benzylic analogs 5a and 5b (picturedbelow) which have different conformational space and flexibilityrelative to direct phenyl substitution of the anomeric oxygen, as withto matched pairs 3a and 3b, show a 2-fold decrease (50% HA titer=60 μM)in potency.

TABLE 10 SAR of simple aryl substitution mannoside library.

Compound R HA 50% Titer (μM) 3a H 30 3b 4-NO₂ 31 3c 3-NO₂ 16 3d 4-NH₂ 323e 3-Me 4 3f 4-Me 8 3g 2-Cl 4 3h 3-Cl 8 3i 4-Cl 32 3j 3-CO₂Me 8 3l3-CO₂H 60 3m 2-CN 6 3n 3-CN 23 3o 4-CN 30 3p 4-OMe 8 3q 2-NHAc 125 3r3-NHAc 12 3s 4-NHAc 8 3t 3-CONH₂ 16 3u 4-CONH₂ 15 3w 4-CH₂CO₂Me 30 43,5-CO₂Me 2

Upon examination of the X-ray structures of α-D-butylmannoside andoligomannose-3 coupled with docking studies of the monophenyl inhibitorsbound to FimH, it was encouraging to observe that additionalimprovements in binding affinity could be achieved by addition of asecond aryl or aliphatic ring system in anticipation of introducing bothadditional hydrophobic and π-π stacking interactions with Tyr-48 andTyr-137 as seen from the directionality of the butyl side chain inbutylmannoside and position of the second mannose residue inoligomannose-3.

To this end, analogs with an additional ring system either directlyattached or fused ring to the parent aryl mannosides of Table 10 wereexplored. It was discovered that a variety of ring systems weretolerated (Table 11) relative to the previously described coumarinanalog 4-methylumbellferyl-α-D-mannoside 6. The most attractiveinhibitor in this series of compounds was the 4′-biphenyl derivative 8ebearing a methyl ester off the meta position of the second aryl ring andshowing a 2-fold improvement relative to 6 displaying an HA titer 1 μM.In order to determine the source of this potency enhancement and aid infurther improvements, a high-resolution X-ray crystal structure of 8ebound to FimH was obtained. Shown in FIG. 22, inhibitor 8e binds in avery complementary “lock and key” fashion to the FimH binding pocket.The mannose ring is making conserved interactions with themannose-binding pocket similar to those described previously while thetwo aromatic rings of the biphenyl moiety exist in a non-planarconformation allowing for a π-stacking and hydrophobic interactions withTyr-48 and other residues encompassing the exterior hydrophobic cleft ofFimH. The π-stacking interaction with Tyr 48 occurs on the opposite sideof the phenyl ring than that engaged by oligomannose-3, which insertsitself into the open “tyrosine gate” formed by Tyr-48 and Tyr-137. Thus,8e engages the “tyrosine gate” in a way that results in alteration ofthe extended FimH binding pocket through closure of the tyrosine gateupon rotation of Tyr-48. This binding mode places the ester of 8e withinH-bonding distance to a salt bridge formed between Arg-98 and Glu-50(FIG. 22). It is noteworthy that the HA 50% titer for Methylαman is >1mM making compound 8e greater than 1000-fold more potent from theaddition of the biphenyl ester.

TABLE 11 SAR of multi-ring system analogs.

HA 50% titer EC₉₀ Compound R (μM) 6

2 7a

8 7b

6 7c

4 7d

2 7e

2 8a

62 8b

6 8c

8 8d

8 8e

1 8f

4 8g

2 8h

2

This observation lead to investigating replacing the mannose withalternate sugars or mimics since the biphenyl portion alone provides asignificant contribution to biological activity presumably from tighterbinding to FimH. It was decided to start by replacing the mannoseportion of 8e with glucose since all chiral centers are identical exceptfor the 3-hydroxyl group adjacent to the anomeric center, being of Rstereochemistry in mannose and S in glucose (Scheme 2). Standard Lewisacid mediated glycosidation gave isomeric mixtures at the anomericcenter favoring the undesired α-isomer, conversely to the cleanformation of the α-isomer seen for the mannosides. Correspondingly,reaction of benzoyl protected α-D-glucose 9 with phenol 10 usingBF₃-OEt₂ followed debenzoylation yielded both the α-D glucoside 11a andα-D-glucoside 11b in a 3:7 ratio. Unfortunately, neither glucosideisomer showed activity in the hemagglutination assay even up to aconcentration of 2.5 mM. Therefore, modification of a single chiralhydroxyl group stereocenter on the mannoside is sufficient tosignificantly decrease biological activity, presumably resulting fromthe inability to tightly bind FimH. Upon analysis of the specificinteractions realized from α-D-mannose bound to FimH,⁴ this hydroxylgroup is making an H-bonding interaction with the N-terminal residue ofFimH and a water molecule contained inside the binding pocket. In anyevent, this finding demonstrates the exquisite specificity of FimH formannose epitopes which has enabled UPEC bacteria to exclusivelyrecognize mannose-presenting cells.

Next, the biaryl portion of the mannoside was optimized by undertakingan extensive SAR evaluation of the second ring through a modifiedToplisstype evaluation of substituents. This focused set of compoundswas designed with some bias toward improving interactions with Arg-98and Glu-50 but also with interest in elucidating the importance of ringelectronic properties as a means to improve the stacking interactionwith Tyr-48. A few initial analogs were prepared using the syntheticroute outlined previously in Scheme 1 but a more convergent synthesiswas developed based on Suzuki coupling of arylbromide intermediate 12 asshown in Scheme 3.

Employing standard Suzuki conditions by reaction of aryl boronic acidsor esters with Pd(Ph₃P)₄ and Cs₂CO₃, 12 was converted to protectedbiphenyl mannosides 13 in good yield. The acylated biaryl mannosides 13were then deprotected as before to generate the final target compounds14 or 15a displayed in Table 12. Di-ester 15a was further functionalizedto di-methyl amide 15b by reaction with dimethylamine or converted todi-acid 15c by basic hydrolysis in methanol. Upon evaluation of HAtiters, the matched pair meta-substituted methyl amide 14a wasequipotent to ester 8e while reverse amide 14b and free acid 14cdisplayed moderately lower potencies of 2 μM and 4 μM respectively. Thelatter result was surprising since these modifications were designed tointroduce an electrostatic interaction with Arg-98 and disrupt theArg-98/Glu-50 salt bridge. On the other hand, sulfonamide 14r hasequivalent potency to carboxamide 14a providing further evidence that ahydrogen bond acceptor is required for optimal potency presumably frominteraction with Arg-98. From analysis of ortho, meta, and para matchedpairs, it was obvious that para-substitution was least preferred foractivity while meta substitution was preferred to ortho substitution.This preference for meta substitution was most pronounced with themethyl alcohols 14g and 14h where the ortho analog 14g was over 5-foldless potent with an HA titer of only 16 μM. It was surmised that thislarge substituent results in an increased rotation of the two rings outof the plane causing a non-productive alignment for interactions withTyr-48. Although a majority of the analogs tested contained electronwithdrawing groups, in general electron donating groups such as themethyl alcohols 14g-i and phenols 14j-l were less active in the HA titerassay. The most potent of the mono-substituted mannosides is meta nitroderivative 14m with a 50% HA titer of 0.5 μM. It is possible that thepartial negative charge on the nitro oxygen atoms allow for an optimizedH-bond acceptor-donor interaction with Arg-98 coupled with the increasedelectron withdrawing ability of the nitro group possibly enhancingstacking interactions with Tyr-48.

TABLE 12 SAR of substituted biphenyl mannosides.

Compound R₁ HA 50% titer EC₉₀ (μM) 14a 3-CONHMe 1 14b 3-NHAc 2 14c3-CO₂H 4 14d 2-CN 2 14e 3-CN 1 14f 4-CN 8 14g 2-CH₂OH 16 14h 3-CH₂OH 314i 4-CH₂OH 6 14j 2-OH 8 14k 3-OH 4 14l 4-OH 6 14m 2-OMe 1 14n 3-OMe 1.514o 2-NHSO₂Me 12 14p 3-NHSO₂Me 2 14q 2-SO₂NHMe 4 14r 3-SO₂NHme 1 14s3-NO₂ 0.5

Perhaps the most unexpected finding from this study was that addition ofanother ester or amide substituted in the other meta position such asdi-ester 15a and di-methyl amide 15b resulted in the two most potentmannoside inhibitors of FimH with activities of 150 nM and 370 nM,respectively. With respect to di-ester 15a this constitutes a 7-foldimprovement in activity relative to mono ester 8e. The di-amide 15b,while slightly less potent, has much improved solubility relative todi-ester 15a. It was hypothesized that the addition of the second esteror amide serves a two-fold purpose for improving activity: First, theelectron withdrawing group results in less electron density of the arylring thus improving π-π stacking interactions with Tyr48. Second, themono-ester (or amide) analog can presumably access conformations inwhich the aryl ring is not in close proximity to Arg-98 whereas the metadi-ester (or di-amide) analog likely exists only in conformations wherethe ester or amide resides in close proximity to Arg-98 resulting inless entropic loss upon binding and a lower energy bound conformationresulting in increased binding affinity and potency.

In order to more accurately determine the relative contributions of bothbinding affinity to FimH versus other compound properties to the potencyseen in the cellular HA titer assay, a high-throughput fluorescencepolarization (FP) (or anisotropy) competitive binding assay wasdeveloped using a fluorescent-labeled mannoside ligand shown in Scheme4. Synthesis was achieved as before using Lewis acid mediatedglycosylation with protected aminoalcohol 17 followed by dual Fmoc andacyl deprotection of intermediate 18 to give free amine 19. Subsequentreaction of 5(6)FAM-OSu with 19 using triethylamine in DMF gave thedesired fluorescently tagged FAM-mannoside 20.

The K_(D) for FAM mannoside 20 was measured to be 0.17 μM while the 50%HA titer was only 125 μM. Twenty mannoside ligands with structuraldiversity and activities were evaluated in the HA titer cell assayranging from 150 nM to 125 μM for their ability to competitively inhibitbinding of FAM 20. Interestingly, it was found that all potentlycompeted for FimH binding having EC₅₀'s less than 0.25 μM but having noclear correlation of EC₅₀ with the activity found in the HA cell assay(Table 13). Although there was over a 60-fold range in cellular potency,there was only about a 5-fold range in binding affinity (FIG. 23). Inaddition, there was also no linear correlation of data with the twoassays. Simple alkyl mannosides have the highest drop in cell activityas demonstrated with butylmannoside having the most dramatic differencewith a 510-fold drop in cell activity relative to binding.

TABLE 13 Binding affinity of selected mannosides in fluorescencepolarization assay. FP Binding HA 50% Compound EC₅₀ (μM) titer (μM)  6(MeUmbman) 0.044 2  8e 0.052 1  8f 0.059 4  3k 0.064 8 14b 0.07 2 14a0.075 1 15a 0.077 0.15 14v 0.078 2  3j 0.084 6 15b 0.087 0.37 Heptylman0.089 15  4 0.091 2 14p 0.097 2 14c 0.099 4  3a 0.114 30  3s 0.122 8  3r0.160 12  3n 0.162 23  5a 0.187 60 Butylman 0.245 125

Interestingly, the most potent biphenyl mannoside series shows a modestcorrelation of binding affinity having a much smaller drop in cellactivity relative to binding. The di-ester 15a and di-amide 15b show thebest correlation with only a 2-fold and 4-fold difference in cellpotency vs. binding. It is important to note that even though these twoanalogs are the most potent in the HA titer cell assay, they have about2-fold less binding affinity in the FP assay than the tightest bindingmannosides. While it is unclear why there is no correlation of bindingto activity in the cell, there is a moderate correlation when compoundswith binned affinities are compared. However, the latter observationcannot explain the non-linear increase in cell activity of 15a and 15brelative to the mono-substituted biphenyl mannosides. Perhapsdifferential binding kinetics including varied on rates and off rates ofmannoside analogs to FimH can provide a plausible explanation for thelatter. It is worth mentioning that the previously reported K_(D) valuesobtained through surface plasmon resonance (SPR) for 6 (MeUmbaman),butylmannoside, and Heptylaman are 20 nM, 151 nM, and 5 nM respectively.While the result for butylmannoside is similar, the FP assay suggestsMeUmbaman is the most potent (44 nM) being 2-fold more potent thanHeptylaman (89 nM). Considering both the lack of correlation betweenbinding affinity and biological activity in the cell plus thevariability seen comparing two separate binding assays, the HA titerassay coupled with the use of structural information is a preferredroute for medicinal chemistry optimization of mannoside FimH ligands.Nonetheless, based on the HA titer cellular data, mannoside 15a is themost potent FimH antagonist reported to date and represents an excellentlead candidate for further optimization of the monovalent mannosidestoward a novel preclinical candidate with tremendous therapeuticpotential for treating urinary tract infections.

The development of multivalent and dendrimeric mannosides has been themajor focus of previous work on FimH antagonists since simplemannosides, while having respectable binding affinity to FimH, show poorcellular activity in the HA assay. It has been demonstrated thatmultivalent mannosides are effective at increasing avidity through aphenomenon termed “cluster effect” resulting in an overall increasedbinding affinity when calculated per mannoside monomer. Therefore, itwas of interest to determine if the improved monovalent mannosides 15aand 15b would produce a similar increase in avidity or cluster effectpreviously reported for dendrimeric mannosides. For syntheticsimplicity, a model system based on monoamide 14a was chosen. Asymmetrical divalent mannoside was designed by connecting two monomersvia an amide based linker to the two biphenyl rings (Scheme 5).Accordingly, dimeric inhibitors based on 14a were synthesized bycoupling carboxylic acid 13c to either diamine2-(2-aminoethoxy)ethanamine or 2-[2-(2-aminoethoxy)ethoxy]ethanamineyielding diamides 21 and 22 respectively using standard HATU couplingconditions with Hunig's base in DMF. The shorter chain analog 21 showeda 50% HA titer of 0.75 μM thus showing no noticeable improvementrelative to 14a, while the longer ethylene glycol linked diamide 22displayed an almost 8-fold increase in activity with an impressive HAtiter of 130 nM. This constitutes a 4-fold increase in avidity relativeto the expected potency of 500 nM based on two monomeric units of 14a.In addition to increased potency, 22 has much improved solubilityrelative to 21 making it an ideal starting point for furtheroptimization. Although these analogs have less potential to be effectiveas oral agents relative to the monovalent mannosides, the divalentmannosides are very useful chemical research tools and can potentiallybe developed into topical or intravenously dosed antibacterial agents.

Example 7. In Vitro Analysis of Mannoside Inhibitors

Three FimH function assays were used to screen mannosides for inhibitoryfunction, and are described below. Selected results for the SAR andtesting of these compounds are presented in Examples 5 and 6, and showthat some synthesized and tested compounds are more potent inhibitors ofHA titer and biofilm formation than anything commercially available andpreviously tested, some by as much as 2 logs.

i. Hemagglutination of guinea pig red blood cells by type 1 piliatedUPEC is dependent upon FimH mannose binding ability. Serial dilutionsallow a quantitative analysis using this assay.

ii. Compounds that decrease hemagglutination by 75% were then tested ina type 1 dependent biofilm assay. First, the ability of mannosidederivatives to prevent UPEC type 1 dependent biofilm formation in thepublished Kolter assay was measured as optimized for the prototypicUTI89 strain used in these assays. Mannoside derivatives that allowgrowth but inhibit biofilm formation at 50% or greater were verified byrepeating the biofilm assay and performing titration curves to determinethe IC₅₀. A more rigorous assay tested the ability of the strongestinhibitory mannoside derivatives to reverse hemagglutination of guineapig red blood cells and to disrupt preformed type 1 dependent biofilms.

iii. A fluorescent polarization assay was developed and miniaturized tomonitor the direct binding of a synthesized fluorescently labeled butylmannose derivative to the FimH receptor as well as competitivedisplacement by other synthetic mannosides. This fluorescentpolarization assay provides a rapid method for monitoring improvementsto receptor affinity during the synthetic process, as well as providingKd binding affinity measurements.

Example 8. Crystallography

In order to further rationally design better mannoside derivatives toinhibit FimH binding the most efficient inhibitors, such as ZFH253, withthe FimH adhesin domain (FimH_(A)) were crystallized to detail theirinteractions with FimH (FIG. 9).

The adhesin domain of FimH (residues 1-175 with a C-terminal 6-histidinetag, hereafter FimHA) was cloned into a pTRC99 plasmid and expressed inE. coli. FimH_(A) was purified from bacterial periplasm by passage overcobalt affinity (Talon; Clontech) and Q Sepharose (GE Healthcare)columns. Protein was subsequently dialyzed against 10 mM MES at pH 6.5and concentrated to 15 mg/ml for crystallization trials. FimH_(A)crystallized in 20% ethanol, 100 mM imidazole pH 8.0, and 200 mM MgCl2.Tetragonal bipyramidal crystals measuring 150×150 pm formed in two daysand did not diffract unless slowly dehydrated over the course of atleast 12 hours. The FimH_(A)-ZFH253 complex was therefore formed bysoaking individual crystals in a stabilizing solution initiallycontaining 15% ethanol, 100 mM imidazole pH 8.0, 200 mM MgCl2, 10%PEG200, 5% glycerol and 1 mM ZFH253. Individual crystals were placed in150 pl stabilizing solution and allowed to dehydrate to one-third theiroriginal volume over the course of 24 hours. Crystals were thenharvested without further cryopretection and plunged into liquidnitrogen. Crystals diffracted to 2.55 Å at beamline 4.2.2 at theAdvanced Light Source. The structure of FimH_(A)ZFH253 was solved bymolecular replacement with the program PHASER using a previously-solvedmodel of FimH_(A) with its methylmannose ligand removed as the searchmodel (PDB ID 1 UWF).

An initial map calculated with Fo-Fc coefficients showed unambiguousdifference density for ZFH253 occupying the mannose binding pocket ofall four copies of FimHA present in the asymmetric unit. These studiesshowed that the mannose portion of ZFH253 engages in conservedinteractions with FimH_(A). ZFH253 buries 293 Å² (calculated with PISA).This places ZFH253 between monomannose, which buries approximately 185Å², and oligomannose-3, which buries 388 Å². Relative to monomannose,most of the additional surface area is buried by the two phenyl groupsof ZFH253, which do not engage the “tyrosine gate” of Tyr48 and Tyr137but orient toward Tyr48 such that the phenyl group most distal to themannose moiety forms a close pi-stacking interaction with Tyr48 (FIG.9). This interaction mimics the aromatic-to-saccharide stacking betweenTyr48 and Man3 of oligomannose-3 that contributes to the high affinityof high-mannose epitopes to FimH in vivo. Additionally, the terminalcarboxyl group of ZFH253 forms two hydrogen bonds with FimH (FIG. 9),offering a structural basis for its 16-fold increase in potency in theHA titer assay over ZFH296, which contains only two unsubstituted phenylgroups. Further, the FimH_(A)/ZFH253 crystal structure suggested severalavenues for rational improvement of mannoside derivatives. First, thereis an ordered water at 2.7 Å from the carboxyl group of the first carbonof the mannose ring that forms close hydrogen bonds with the N-terminus,the main-chain carbonyl of Phe1, and the epsilon oxygen of Gln133.Extension of this carboxyl group by two bond lengths would allow themannoside to engage these interactions. There is a second ordered water4.7 Å from an ortho carbon of the first phenyl group of the mannosidethat could be coordinated by a hydrogen bond donor. Importantly, theFimH_(A)/ZFH253 crystal structure serves as proof of concept thatcomputational docking may serve as a useful tool in rational design ofrelated mannosides. The conformation of ZFH253 seen in the crystalstructure is essentially identical to that of a lowest-energy conformergenerated by OMEGA2 and docked into a previous crystal structure of FimHby FRED (OpenEye, Inc.). This approach will speed the design of furthercompounds, with additional crystallography for the exploration ofscaffolds other than the biphenyl group of ZFH253.

Example 9. Inhibition of In Vivo Functions of FimH

Based on the extensive elucidation of the UPEC pathogenic cascade,critical nodes in pathogenesis that mannosides would target and therebyhave powerful and potent therapeutic ramifications were identified. Thefirst step in the UPEC pathway involves FimH-mediated colonization ofthe bladder epithelium via the recognition of mannose receptors.Bacterial invasion into superficial bladder umbrella cells can thenensue. By preventing bacterial attachment, mannosides will also inhibitthe subsequent invasion of bacteria into the bladder epithelial cells.To test this, a well-established gentamicin protection assay that allowsthe determination of the luminally bound fraction versus theintracellular fraction was utilized (FIG. 10). In the presence ofmannosides ZFH177 and ZFH253 there was a significant reduction inintracellular bacteria at 1 hour post-infection arguing that themannosides are disrupting specific type 1-mediated binding required forsubsequent invasion. It is not clear why a reduction was not observed inthe luminal UPEC population. This may be due to contamination frombacteria in the urine or bacteria that are non-specifically associatedwith the epithelium. In any case, blocking invasion blocks the abilityof the bacteria to rapidly expand in numbers. This is due to the abilityof the mannosides to block the IBC mechanism.

After invasion, UPEC are able to rapidly replicate to form IBCs, thusdramatically expanding in numbers. After maturation of the IBC, bacteriaare able to disperse from the biomass and spread to neighboring cells toexpand the buildup in bacterial numbers via next generation IBC. Theability of mannosides to block IBC formation was evaluated based on LacZstaining of whole mount bladders, a protocol developed to enumerateIBCs. Whole bladder titers were also determined by quantifying colonyforming units (CFUs) at 6 hours post-infection and later time points astest compounds were further developed. In the presence of mannosidesZFH177 and ZFH253 there was a significant decrease in IBCs observed 6hours post-infection at concentrations as low as 0.1 mM and almost noIBCs observed at 1 mM Mannoside (FIG. 11A). A significant decrease wasalso seen in CFUs at 6 hours post-infection with 1 mM ZFH177 and ZFH253(FIG. 11B). These results strongly argue that the mannosides are able toinhibit first round IBC formation by preventing FimH-mediated bindingand invasion into the bladder epithelial cells. The success of theseexperiments suggested that these compounds would have dramatic potentialas potent therapeutics for treating UTI, the most common bacterialinfection in highly industrialized countries.

Example 10. Pharmokinetics/Bioavailability/Toxicity

Pharmokinetic, bioavailability and evaluation of toxicity studies willbe performed to elucidate the effectiveness of synthesized mannosides intreating UTI. Using these studies we will identify: i. the kinetics bywhich the mannoside is excreted in the urine, ii. the dosageadministered to the mouse versus the amount that reaches the urine toobtain 1 mM and 0.1 mM concentrations (determined effective throughabove results), iii. the length of time the mannoside remains in theurine after one dose and iv. the presence of mannoside within thetissue, namely the bladder. The most effective way of delivering themannoside (oral, IP or tail vein) with no toxicity to the animal will bedetermined. Toxicity will be evaluated based on animal survival andweight loss/gain of treated animals relative to non-treated animals.Urine was evaluated for mannoside presence and concentration usingLC-MS. The delivery method, dose and timing, which gave the highestlevels in the urine were then used in treatment trials.

Example 11. Mannosides as Inhibitors of UTI

Once the proper delivery method, dose and timing will be determined,female mice will be pre-treated with mannoside (two doses) prior toinfection (using our well-developed mouse model of UTI) and treated withmannoside throughout the course of infection to maintain efficaciouslevels within the urine. The potency of the mannoside in preventing UTIbased on CFUs in the bladder will be assessed at 6 hours, 48 hours and 2weeks post-infection and IBC formation at 6 hours post-infectioncompared to untreated mice.

The ability of mannosides to treat established infections will be alsotested. Mice will be treated with mannoside at 24 hours post-infectionand the effect on bacterial clearance will be assessed by determiningCFUs in the bladder 24 and 48 hours and two weeks post-mannosidetreatment. The last set of experiments mimic a potential situationwithin the clinic.

These experiments will show that inhibition of binding of the FimHadhesin to the bladder epithelium can eliminate infection. The aboveexperiments also establish that the mannosides block the formation ofQIRs, which proves their efficacy in reducing the predisposition of thehost to suffer a recurrent infection. Due to increasing antibioticresistance and high rates of recurrence, these compounds introduce anentirely new means of treating microbial infections, using ananti-virulence agent instead of an antimicrobial.

Example 12. Investigation of Dual Therapy with Adhesion and PilusAssembly Inhibitors and/or Antibiotics

Due to the critical nature of type 1 pili in UPEC pathogenesis, inaddition to the work to develop high affinity inhibitors of FimH bindingdescribed above, the extensive structural and functional knowledge ofthe mechanism of pili assembly by the chaperone/usher pathway (seebiosketch) was used to develop efficient inhibitors of pilus biogenesis(“pilicides”) (FIG. 12). The most efficient pilicides show greater than90% inhibition of hemagglutination and type 1 dependent biofilm atconcentrations in the low micromolar range (FIG. 13). Further, thestructural basis by which pilicide block pilus assembly were identified.These compounds were rationally designed based on a 2-pyridone scaffoldthat has affinity for beta-sheet structures. One of the best pilicidesto date, FN075, was able to achieve significant attenuation of virulenceas measured by CFUs and IBC formation (FIG. 14).

Using pilicides and mannosides in combination would potentially give a“double hit” to this critical type 1 pilus system, potentially resultingin synergy by inhibiting pre-existing type 1 pilus function whileeliminating the ability to produce new pili. Combination therapy usingthe most effective mannoside inhibitors and pilicides with antibioticswere tested. In one experiment, groups of mice were treated with ZFH56mannoside treatment only, with antibiotic treatment only, or with a dualtreatment (mannoside+antibiotic). One group of untreated mice was usedas a control (FIG. 15).

For mannoside treatment, bacteria were incubated with 1 mM ZFH56 priorto inoculation. Every 24 h for 3 days, mice were transurethrallyinoculated with 1 mM ZFH56. For antibiotic treatment, mice wereinoculated with UTI189 and immediately given antibiotics in the drinkingwater for 3 days. Antibiotics used were trimethoprim-sulfamethoxazole(SXT) at 54 and 270 mg/ml, respectively. For dual treatment, bacteriawere incubated with 1 mM ZFH56 prior to inoculation. Every 24 h for 3days, mice were transurethrally inoculated with 1 mM ZFH56.Additionally, mice were immediately given antibiotics in the drinkingwater for 3 days as in the antibiotic only treatment. Urine wascollected every day for 13 days. At 2 weeks, mice were sacrificed andbladders were titered.

Dual treatment showed significantly lower levels of bacteria in thebladder than antibiotics alone (FIG. 16).

Example 13. Bacterial Adhesion—A Source of Alternate Antibiotic Targets

Introduction

More than a century ago, the discovery of Penicillin marked asignificant, albeit not immediately recognized, advance in the field ofmedicine. By the middle of the 20^(th) century this naturally occurringfungal antibiotic had single-handedly vanquished the biggest wartimekiller—infected wounds. Just four years after the mass production ofpenicillin began in 1943, resistant microbes started to appear beginningwith Staphylococcus aureus, followed shortly thereafter by Streptococcuspneumoniae and Neiserria gonorrheae. Today this list includes antibioticresistant Enterococcus, Salmonella, Mycobacterium tuberculosis andEscherichia coli, to name just a few. The bacterial infections whichcontribute most to human disease are also those in which emerging andmicrobial resistance is most evident: diarrheal diseases, respiratorytract infections, urinary tract infections, meningitis, sexuallytransmitted infections, and hospital acquired infections. Thus, there isdire need for new therapeutics to save the lives and ease the sufferingof millions of people.

Rising Antibiotic Resistance

The antibiotics that are available today are primarily variations on asingle theme-bacterial eradication based on inhibition of essentialmolecular processes required for bacterial growth (bacteriostatic) orcellular maintenance (bactericidal). Some target cell-wall biosynthesis,whereas others inhibit protein synthesis or DNA replication. These lifeor death treatments put selective pressure on the bacteria to adapt ordie, selecting for antibiotic resistance. Second generation drugs weredeveloped to combat the resistances that arose, however, they employedthe same general mechanism of action. In addition, while some lastresort drugs have minimal resistance thus far, they are much moreexpensive, often are more toxic and typically unavailable to manycountries around the world. Consequences of treatment failure due toantibiotic resistance are high with greater morbidity, mortality andtransmission of the resistant organism. Even if pharmaceutical companiesincreased efforts to develop next generation replacement drugs,bacterial evolution is outpacing drug development for these classes ofantibiotics. We are nearing an end to the seemingly endless flow ofantimicrobial drugs. We are now faced with a long list of microbes thathave found ways to circumvent different structural classes of drugs andare no longer susceptible to most, if not all, therapeutic regimens. Newtactics and weapons are needed to combat bacteria that are, owing toevolution and selection, moving targets.

Alternative Antibiotic Targets

Targeting bacterial virulence is an alternative approach to thedevelopment of new classes of antimicrobials that can be used tospecifically target and disarm pathogens in the host, while leavingcommensal bacteria unperturbed. Prevention of the activity of virulencefactors will render the bacteria less able to colonize and givebeneficial symbionts and the host immune system time to eradicate thedisease causing pathogen prior to colonization or facilitate clearing ofan established infection. Furthermore, stripping microorganisms of theirvirulence properties without threatening their existence may offer areduced selective pressure for drug-resistant mutations and provideincreased potency. Upon exposure to the host, bacteria respond by theproduction of an arsenal of virulence factors to combat host immuneresponses and facilitate persistence in their desired niche. There aremany examples of virulence factors that represent potential therapeutictargets. Polysaccharide capsules prevent phagocytosis. Siderophoresfacilitate iron acquistion. Flagella promote motility and chemotaxis.Some bacterial species secrete toxins that alter and disrupt theeukaryotic host cell resulting in disease. Yet another strategy employedby bacteria is the production of a molecular syringe, known as a typethree secretion system, that injects effectors into the host cellcausing disease. Another fundamental virulence property of nearly allmicrobes is their ability to adhere to host cells. This step istypically crucial in pathogenesis and without it bacteria are morelikely to be eradicated by the host. Bacterial adhesins typicallymediate attachment to a specific receptor with stereochemicalspecificity, a process that is thought to determine the host and tissuetropisms of a pathogen. While many virulence factors may representattractive therapeutic targets, this review will focus on two strategiesto develop anti-virulence therapeutics based on targeting adhesive piliusing uropathogenic E. coli (UPEC) as the model system. The firststrategy depends on competitively inhibiting bacterial binding to hostcells through addition of exogenous carbohydrates that mimic hostligands. The second strategy targets the mechanism by which adhesivepili are assembled thus inhibiting bacterial adhesion to host cells.Thus, both strategies could be used in synergy: the first compoundprevents bacterial attachment and colonization by occupying the bindingpocket of the bacterial adhesin while the second compound enters thecell and prevents future pilus production. Such a two-pronged approachcould eliminate adherence to host cells and promote clearance of thebacteria.

Pili in Bacteria

Pathogens are capable of presenting multiple adhesins that can beexpressed differentially to permit binding in specific sites and atparticular times over the course of a complex infectious cycle. Toachieve this, many bacterial species possess long filamentous structuresknown as pili or fimbriae extending from their surfaces. Pili areextracellular polymers that also have been shown to play a role ininvasion, biofilm formation, cell motility, and protein and DNAtransport across membranes. Pilus formation is common to many pathogenicbacteria, both Gram-negative and Gram-positive pathogens. Despite thediversity in pilus structure and biogenesis, assembly mechanisms amongGram-negative and Gram-positive bacteria are conserved within eachgroup. Gram-positive pili are formed by covalent polymerization of pilinsubunits in a process that requires a dedicated sortase enzyme. Incontrast, Gram-negative pili are typically formed by non-covalenthomopolymerization of major pilus subunit proteins. This review willfocus on a major family of adhesive pili in Gram-negative bacteria thatare classified by their mechanism of assembly; the so-calledchaperone/usher (CU) pili (Table 14).

In Gram-negative bacteria, one of the best-characterized pilus assemblysystems is the CU pathway. Hundreds of operons encoding CU systems havebeen reported in a plethora of pathogenic organisms with as many astwelve encoded by a single organism. Examples of CU-assembled adhesivevirulence fibers include: Type 1 and P pili predominately on UPEC thatcause urinary tract infection (UTI), S pili of E. coli strains thatcause sepsis, meningitis and UTI and Hif pili of Haemophilus influenzawhich causes otitis media. The causative agents of whooping cough(Bordetella pertussis), gastroenteritis (Salmonella typhimurium), andpneumonia (Klebsiella pneumoniae) also assemble fibers through the CUpathway. Additionally there are ‘non-pilus adhesins’ assembled by the CUpathway that are generally homopolymers composed of a single proteinsubunit, like the Afa/Dr family of adhesins of E. coli and the polymericF1 capsular antigen of Yersinia pestis. The ubiquitous nature of thispathway paves the way for a potentially broad-spectrum anti-virulencetherapeutic that disrupts the assembly of the pilus fibers thuseliminating bacterial colonization.

Pili in UTI

Of the CU pathway assembled pili, P and type 1 pili are the mostextensively characterized. Type 1 pili are expressed throughout theEnterobacteriaceae family but have been shown to be essential for UPECin colonization of the urinary epithelium. UPEC is the leading causativeagent for greater than 80% of UTIs which is one of the most commonbacterial infections in industrialized countries. UTIs are a significantburden, resulting in more than 8 million outpatient visits per year inthe United States and expended costs of US $3.5 billion annually forevaluation and treatment. Women are the most frequent sufferers—a femalehas a 60% chance of suffering from at least one UTI in her lifetime.Additionally, up to 44% of women who present with an initial episode ofUTI will experience recurrence. While the increased likelihood ofrecurrence it not entirely understood, data suggests that bacteria canpersist within bladder tissue despite antibiotic treatments, and mayserve as bacterial reservoirs for recurrent infections.

Type 1 pili are expressed by virtually all UPEC isolates. The FimHadhesin at the tip of type 1 pili achieves specific binding to hostcells within the urinary tract. FimH specifically binds mannose groupsthat abundantly decorate uroplakins on the luminal surface of thebladder. A deep, negatively charged pocket in the N-terminalreceptor-binding domain of FimH accommodates the mannose withstereochemical specificity. Once bound, a pathogenic cascade isinitiated that involves several distinct phases as examined in themurine cystitis model and human UTIs. Numerous innate defenses areactivated early in an attempt to stem off the infection. These defensesinclude, cytokine induction followed by inflammation, iron sequestering,exfoliation of the colonized epithelial cells and sheer forces elicitedby urine flow. Even if UPEC successfully invade into a bladderepithelial cell, the bladder cell has been shown to be able to expelUPEC, potentially serving as an additional innate defense. However, ifUPEC are able to escape into the cytoplasm of superficial umbrellacells, they are able to rapidly replicate to form tightly packedintracellular bacterial communties (IBCs) with biofilm-like qualities.Within the urothelial cells, the bacteria are protected from the flow ofurine and host defenses. Eventually UPEC detach from the IBC, disperseand initiate new rounds of IBC formation. This represents a mechanism bywhich a single invasion event dependent on type 1 pili adhesion can leadto rapid bacterial expansion and colonization of the urinary tract. IBCformation occurs in the acute states of infection. One long-term outcomeof infection is that UPEC are also able to form a quiescentintracellular reservoir (QIR) within the bladder. Bacteria in the QIR donot rapidly replicate, but remain dormant, hidden from the immune systemand antibiotics. They are then able to reestablish an infection at latertimepoints post-treatment resulting in a recurrence. Due to thisalternative pathway of continuous colonization, UTIs are more frequentlybeing identified as chronic infections. Detailed analysis of the UPECpathogenic cascade has identified several potential targets to inhibitvirulence, such as siderophores, capsule and flagella. However, type 1pili represent a particularly attractive drug target because they areubiquitous among UPEC, and are required to initiate a pathogenic cascadethat evades the host immune system and can lead to acute, chronic,persistent or asymptomatic infection. Disruption of type 1 pili functioncould break the cycle of chronic infection.

New Drug Development Strategies to Inhibit Pilus Mediated Function

Carbohydrates of various forms are abundantly expressed on the animalcell surface. Microbes have taken advantage of this property and evolvedwith the ability to adhere to sugar receptors in an organ-specificmanner for colonization and infection. Sugars are commonly involved incell recognition and communication playing important roles in microbialadherence and uptake, colonization and biofilm formation, and invirulence. Since bacterial adhesion to host cells is extremely specific,exogenous sugars can block binding to carbohydrate receptors andcompetitively displace or inhibit bacteria from attachment to cells. Theability to impair bacterial adhesion represents an ideal strategy tocombat bacterial pathogenesis because it targets an important early stepin the infectious process, and would also be suitable for use as aprophylactic to prevent infection. In the case of UPEC, knowledge of thestructural basis of how FimH recognizes the mannose receptor, has leadto the development of a ligand-based antagonist, termed mannoside, thatmimics the natural receptor for FimH but with increased affinity andavidity. Mannosides effectively block the adhesive properties of type 1pili by inhibiting bacterial colonization, invasion, IBC formation anddisease. Furthermore, the sequence of approximately 300 fimH genes ofclinical UPEC isolates showed that the mannose-binding pocket isinvariant. Thus mannosides are a powerful new class of therapeutics thatcould have a potent therapeutic effect by preventing UPEC pathogenesisand disease. This approach can be readily extended to other adherentorganisms by tailoring the anti-adhesive compounds to antagonize theirspecific receptor-ligand interactions (FIG. 17). Indeed, sugars designedto target adhesive structures of organisms such a Pseudomonasaeruginosa, Stapyhlococcus and Streptococcus have already provedefficacious at inhibiting adherence in vitro.

The goal of the second strategy is to interrupt pilus assembly, which,like the first strategy, blocks pilus-mediated adhesion and subsequentdisease. To disrupt pilus assembly, one must first understand themechanisms employed by bacteria to form fibers. Chaperone-usher mediatedassembly prevents premature subunit aggregation and facilitates theordered secretion, folding and assembly of hundreds of thousands ofsubunits into fibers on the surface of a bacterium.

Components of the pilus are secreted through the general secretorypathway into the periplasm. Once in the periplasmic space, a specificchaperone binds each pilus subunit to facilitate proper folding andprevent premature assembly of subunits. Each subunit consists of six ofthe seven strands of a standard immunoglobulin (Ig)-fold and anN-terminal extension (Nte) (FIG. 18A). In order to form a stablestructure prior to incorporation into the growing fiber, the chaperonedonates structural information to each subunit, its G1 β strand, tocompensate for the missing seventh strand of the subunit in a processcalled donor strand complementation (DSC) (FIG. 18B). DSC preventsaggregation of the subunits prior to fiber formation. Thepilin/chaperone complex is then delivered to the usher, which is a porein the outer membrane and serves as a platform for pilus assembly.During pilus assembly, the free Nte of one subunit displaces thechaperone bound to another subunit and serves as the seventh strand ofthe Ig-like fold in a process called donor strand exchange (DSE) (FIG.18C). DSE leads to the polymerization of the fiber extending from thebacterium. Since chaperone-usher systems are necessary for the assemblyof extracellular adhesive organelles in a wide range of pathogens,inhibitors may serve as broad-range anti-virulence therapeutics, anattractive feature that would enhance the marketability of an effectivedrug.

Pilicides are a class of pilus inhibitors that target chaperone functionand inhibit pilus biogenesis. A recent study (Pinkner J S et al: PNAS(2006) 103(47):17897-17902) identified a new class of pilicides based ona bi-cyclic 2-pyridone chemical scaffold that inhibit pilus assembly bybinding to the chaperone and disrupting the chaperone-usher interaction.As a result, pilicides inhibit pilus dependent activities such ascolonization, invasion and biofilm formation. Biofilms are structuredcommunities of microorganisms encapsulated within a self-developedpolymeric matrix that defends them against antibiotics and the hostimmune system. They are of great medical importance, accounting for over80% of microbial infections in the body. Pilicides target regions on thechaperone that are highly conserved among chaperone-usher pathways andare thus able to inhibit assembly of multiple pilus systems (type 1 andP pili of E. coli) (FIG. 17). Pyridinones also avoid the severedrawbacks observed for peptide-based drugs; poor absorption after oraladministration, rapid enzymatic degradation, and quick excretion.

Another distinct mechanism of adhesive fiber assembly amongGram-negative bacteria is fibers assembled by the extracellularnucleation/precipitation pathway (curli). Curli are proteinaceous fibersfound on enteric bacteria such as E. coli and Salmonella spp. Despitecurli's unclear role in pathogenesis, their biochemical and structuralproperties resemble eurkaryotic amyloid fibers found inneurodegenerative diseases, such as Alzheimer's, Parkinson's, and priondiseases making them an ideal model system to study amyloid biogenesis.Curli are heteropolymers that consist of a major subunit, CsgA, andminor subunit, CsgB. CsgA and CsgB are secreted into the extracellularmilieu via the outer membrane pore, CsgG. With the help of twochaperone-like proteins, CsgE and CsgF, CsgA is nucleated into a fiberby CsgB. Curli fibers have been implicated in biofilm formation,environmental survival and resistance to sanitizing agents. A recentstudy (Cegelski L et al: submitted) showed that derivatives of the2-pyridone compounds mentioned above were able to inhibit curli assemblygiving them the name curlicides. The lack of curli fibers preventedcurlidependent biofilm formation and reduced infection load in vivo asmeasured by the murine model of UTI. Pilicides and curlicidesselectively disrupt a protein-protein interaction required for thebiogenesis of a bacterial virulence factor instead of targeting aprocess essential for survival.

Employing both anti-virulence strategies, inhibition of receptor bindingand disruption of pilus assembly, could result in very potentanti-adhesive therapeutics. For example, synergistic effects could beobtained by inhibiting preexisting type 1 pilus function throughmannosides while also eliminating the ability to produce new pilithrough pilicides. Furthermore, if infection is not entirely controlledby the two-pronged approach, antibiotics can be added, however, at muchlower concentration to reduce the opportunity to develop resistance.Type 1 pili are an essential virulence factor for colonization of theurinary tract and are the most extensively studied chaperone-usherfimbrial system. Thus, they have been described in this review as anexcellent target resulting in the development of new prototypictherapeutics to treat UTIs. This new drug class could translate into amultitude of other therapies for Gram-negative infections.

Conclusion

While this review focuses on the development of therapeutics to inhibitadhesion, there are many other virulence factors employed by bacteria tocolonize and persist within the host. The general strategy of inhibitingvirulence factors rather than essential growth factors changes theparadigm of drug development and reduces the evolutionary pressure forbacteria to develop resistance. Bacteria can survive in the presenceanti-virulence drugs, but will be eliminated by the host immune systemprior to establishing a foothold. Furthermore, since a complex virulencemechanism is targeted, if mutants arise, they will most likely beavirulent. If the host immune system cannot resolve any residualinfection, it is possible to couple anti-virulence therapeutics withtraditional antibiotics although at lower doses thus reducing resistancepotential. A commitment to developing therapeutics that target virulencerequires a serious change in our perspective for treating infectiousdiseases and increased efforts should be focused on bringing these newtherapeutics from bench to bedside.

Example 14. Orally Active Mannosides Subvert Antibiotic Resistance of E.coli in Bladder Infection

In this example, therapeutic utility of mannosides that are competitive,high-affinity inhibitors of FimH mannose binding activity is analyzed,as measured by biofilm formation, type 1 pilus expression and bladdercolonization, invasion and IBC formation. The mannose binding site ofFimH consists of a deep acidic pocket surrounded by a ridge ofhydrophobic residues. The acidic amino acid residues of the bindingpocket are invariant in all UPEC and mutational studies showed thataltering any of these residues abolishes binding to mannose andattenuates virulence. From the rational design and optimization ofmannosides, several potent inhibitors of FimH-dependent hemagglutinationwere identified, including ZFH-1101, ZFH-1177, ZFH-1253, ZFH-2050 andZFH-2056 (FIG. 19a ). These mannosides were evaluated first for theirability to inhibit biofilm formation or disrupt preformed biofilms.Biofilm inhibition was evaluated on FimH-dependent UTI89 biofilms grownon PVC in LB at room temperature. The median inhibitory concentration(IC₅₀) values for each of the compounds were found to be in the lowmicromolar range. Compounds ZFH-1253 and ZFH-2056 were the most potentbiofilm inhibitors with IC₅₀ values of 0.94 and 0.74 μM, respectively(FIG. 19b ). Biofilm disruption was evaluated by the addition ofmannosides to preformed 24 h biofilms followed by growth for anadditional 16 h. All five mannosides were shown to facilitate dispersalof the preformed biofilm. The IC₅₀ values for dispersal were higher thanthose concentrations needed to prevent biofilm formation, however theefficacy trend remained the same (FIG. 19c ). Visualization ofmannoside-treated PVC biofilms by confocal microscopy showed thatZFH-2056 treated biofilm lacked continuity as demonstrated by holes inthe biofilm, and it lacked the tall mushroom-like structures observed inuntreated biofilm (FIG. 19 d,e). Biofilm causes many antibiotictreatment failures since antibiotics are unable to penetrate its densematrix. These results demonstrate that mannoside can prevent biofilmformation and dissolve preformed biofilms arguing that mannosides mayaugment antibiotic efficacy by altering bacterial community behavior.

To test the efficacy of the mannosides in vivo, UTI89 expressing type 1pili were pre-incubated with mannosides, inoculated into mice and IBCswere quantitated at 6 hours post-infection (hpi). The efficacy ofcompounds ZFH-1177, ZFH-1253, ZFH-2050 and ZFH-2056 in inhibiting IBCformation correlated with their potency observed in the in vitro assays.The most significant reduction in IBCs occurred with as little as 10 μMZFH-2050 or ZFH-2056 (FIG. 20a ). These studies identified ZFH-2056 asthe most potent compound tested in preventing colonization.

Pharmacokinetic (PK) studies with ZFH-2056 evaluated in vivo dosingconditions after intraperitoneal (IP) injection or oral (PO) gavage.ZFH-2056 was dosed IP and compound levels in the urine were evaluated atseveral timepoints by HPLC using mass spectrometry (MS) detection. Dosesof 5 mg/kg and 10 mg/kg resulted in concentrations of nearly 5 mM in theurine 30 min after treatment (FIG. 20b ). Eight hours after dosing,ZFH-2056 remained at levels equal to or slightly below the IC50 (0.74μM) as determined by biofilm inhibition. Since oral dosing is thedesired route of delivery, we next evaluated the concentration of drugafter 100 mg/kg PO of ZFH-2056. The latter resulted in a 10 fold lowerinitial concentration of ZFH-2056 versus IP but, significantly, urinelevels were 10-fold higher at 8 hours post-dosing. It is noteworthythat >95% of drug cleared to the urine is intact with the remaining <5%being the hydrolysis products, D-mannose and phenol.

The efficacy of in vivo mannoside treatment was evaluated after dosinganimals with mannoside either IP or PO 30 min prior to infecting withUTI89. At 6 hpi the bladders were removed and total bacterial CFUs werequantitated. In both the IP and PO treated cohort, there was asignificant drop in bacterial counts demonstrating the efficacy ofmannoside in reducing overall colonization of the bladder (FIG. 20e ).Furthermore, IBC quantification showed a significant reduction in IBCsin the mice that were pretreated with mannoside by either the IP or oralroute (FIG. 20f ). To test whether mannoside reduced IBC formation byblocking UPEC invasion into the bladder tissue, gentamicin treatmentassays were performed. Gentamicin efficiently kills extracellular UPECbut is unable to penetrate tissue and thus intracellular bacteriasurvive gentamicin treatment 18. In the ZFH-2056-treated mice,gentamicin treatment of the bladders eliminated all CFUs (FIG. 20g ). Inbladders from untreated mice, 10³-10⁴ CFUs remained after gentamicintreatment. These results argued that mannoside effectively preventedUPEC penetration into the bladder tissue. Confocal microscopy confirmedthese results. Bladders of the untreated cohort showed normal, robustIBC formation (FIG. 20c ) whereas IBCs were rarely seen in the mannosidetreated mouse bladders but bacteria were observed on the bladderepithelial surface (FIG. 20d ). These results demonstrate thatmannosides prevent bacterial invasion into the bladder tissue andsignificantly reduce infection in the bladder.

The first-line treatment of choice for UTI has long been a 3-day courseof TMP-SMZ. However, resistance to TMP-SMZ is on the rise. Furthermore,humans have a 38% carriage rate of TMP-SMZ resistant (TMP-SMZR) strains,and these numbers are even higher in low resource countries with a 94%carriage rate. TMP-SMZ is known to concentrate in the urine. Therefore,it was reasoned that by preventing bacterial invasion into the bladdertissue, mannoside may have an anti-virulence synergism with TMP-SMZ andmay circumvent the TMP-SMZ resistance problem. This was tested in micegiven TMP-SMZ for 3 days prior to inoculation with either UTI89 orTMP-SMZR strain, PBC-1. Mice were either untreated or IP treated withmannoside ZFH-2056 30 min prior to inoculation with bacteria. Afterinoculation with UTI89 or PBC-1, bacterial CFUs were quantitated 6 hpi.Upon treatment with only ZFH-2056 there was a significant drop inbacterial load of both strains in the bladder. As expected, treatmentwith TMP-SMZ alone resulted in a significant drop in bacterial load inthe UTI89-infected mice but had no effect on PBC-1, since it isresistant to TMP-SMZ. In the dual treatment group there was asignificant drop in bacterial CFUs compared to mannoside alone orTMP-SMZ alone for both strains which was most pronounced for PBC-1 (FIG.21 a). The observation that TMP-SMZR strain PBC-1 was able to succumb toantibiotic treatment in the presence of mannoside suggested that eithermannoside increases the efficacy of TMP-SMZ killing or that it exposesthe bacteria to concentrations at or above the MIC necessary forkilling. Mannoside was not found to increase the efficacy of TMP-SMZkilling when bacteria were grown in varying concentrations of TMP-SMZwith and without 100 pM of ZFH-2056 (FIG. 21 b). This analysis revealedthat the TMP-SMZ resistant strain had a MIC of 256 and 1280 pg/ml,respectively. Furthermore the HA titer of PBC-1 after growth in varyingconcentrations of TMP-SMZ showed that type 1 pili function was notaffected by antibiotic (data not shown). It has long been known that TMPconcentrates in the urine, and this serendipitous feature is one reasonTMP-SMZ has been the antibiotic of choice for UTI over the last severaldecades. Using MS, the concentration of TMP-SMZ was measured in theurine of mice after 3 days of treatment with 54 μg/ml and 270 μg/ml TMPand SMZ, respectively. TMP concentrations were determined to be9.95+/−4.36 mg/ml. SMZ was present at 67.17+/−32.51 μg/ml. These resultsshow that by preventing bacterial invasion, mannoside keeps UPECextracellular thus exposing them to TMP concentrations in the urine thatare well above the MIC of the UPEC strain. This enables the killing ofbacteria that clinically are resistant to TMP-SMZ. Thus, the invasion ofthe TMP-SMZR UPEC strain PBC-1 into bladder tissue protected it from theexposure of TMP-SMZ concentrations of antibiotic necessary for killing.However, mannoside prevented access of the bacteria to the intracellularcompartment and thus kept the bacteria extracellular where TMP-SMZconcentrates to high levels in the urine, resulting in their succumbingto killing by the antibiotic. These results highlight the importance ofthe intracellular pathway in bacterial persistence. Not only do thebacteria escape the immune system in their intracellular niche, they arealso able to escape exposure to effective antibiotics.

Methods for Example 13

UTI189 biofilm was grown in LB+/−mannoside for 24 h at 22° C. in PVCplates and quantitated using crystal violet. UTI189 biofilm for confocalmicroscopy was grown in LB for 24 h at 22° C. on PVC coverslips.Mannoside was then added and biofilm was grown for an additional 16 h.Coverslips were then washed in PBS, fixed in 2% paraformaldehyde andstained with SYTO9 prior to confocal microscopy. For all animalexperiments UTI189 or PBC-1 was grown 2×24 h statically in LB at 37° C.and inoculated at a dose of 1×10′ bacteria in 50 μl. All mice used werefemale C3H/HeN (Jackson). For the pretreatment studies, bacteria wereincubated with mannoside for 20 min prior to inoculation into the mouse.For IP dosing, 50 μl of 2 mg/ml (5 mg/kg) or 4 mg/ml (10 mg/kg) ZFH-2056in PBS was injected into the mouse 30 min prior to inoculation ofbacteria. For oral dosing, 100 μl of 20 mg/ml (100 mg/kg) ZFH-2056 in 8%DMSO was inoculated with a gavage needle 30 min prior to inoculation ofbacteria. Mass spectrometry was used to quantify urinary mannoside orTMP-SMZ concentrations. For CFU counts, bladders were harvested at 6 hpiand placed in 1 mL PBS. Bladders were then homogenized, diluted andplated on LB. After growth at 37° C. overnight, bacterial counts weredetermined. LacZ staining and gentamicin protection assays wereperformed at 6 hpi. For antibiotic experiments, mice were given TMP-SMZin the drinking water at a concentration of 54 μg/ml and 270 μg/ml,respectively. Water was changed daily with fresh antibiotics. Standardgrowth curve assays and hemagglutination assays were performed withTMP-SMZ. All statistical analysis performed was a two-tailedMann-Whitney U test.

Bacterial Strains.

UTI189 is a prototypical cystitis isolate of serotype O18:K1:H7. PBC-1is a TMP-SMZR strain of serotype OX13:K1:H10 isolated from a 59 year oldasymptomatic female with a history of recurrent UTI and diagnosis ofprimary biliary cirrhosis.

Synthesis of Mannosides.

Following the procedure outlined in Han et. al. 2010 (J Med Chem 53(12), 4779-4792), ZFH-2056 was synthesized on a multi-gram scalewith >98% purity by HPLC and NMR.

Biofilm Assay.

UTI189 was grown in LB broth in wells of PVC microtiter plates at 23° C.in the presence of individual mannosides at varying concentrations.After 48 h of growth, wells were rinsed with water and stained withcrystal violet for quantification as described. For biofilm disruptionactivity in PVC plates, UTI189 was grown in LB broth in wells of PVCmicrotiter plates at 23° C. After 24 h of growth, mannoside was addedand biofilms were grown for an additional 16 h. Wells were then rinsed,stained with crystal violet and quantified. For biofilm disruptionactivity on PVC coverslips, UTI189 was grown in LB broth in 50 mLconicals containing PBC coverslips at 23° C. After 24 h of growth, 0.3μM ZFH-2056 was added and biofilm was grown for an additional 16 h.Coverslips were then rinsed, fixed with 2% paraformaldehyde (v/v),stained with SYTO9 (1:1000 in PBS; Molecular Probes) and observed with aZeiss LSM410 confocal laser scanning microscope under a 63× objective.

Animal Infections.

Bacteria were grown under type 1 pili-inducing conditions (2×24 h at 37°C. statically in LB). The bacteria were harvested and resuspended to anOD₆₀₀ of 0.5 in PBS. Eight-week-old C3H/HeN (Harlan) female mice wereanesthetized by inhalation of isoflurane and infected via transurethralcatheterization with 50 μl of the bacterial suspension, resulting in1-2×10′ inoculum. At 6 hpi, mice were sacrificed by cervical dislocationunder anesthesia and the bladders were immediately harvested andprocessed as described below. All animal studies using mice wereapproved by the Animal Studies Committee of Washington University(Animal Protocol Number 20100002).

Enumeration of Bladder IBCs.

For bacterial pretreatment experiments, mannoside was added to thebacterial suspension and incubated for 20 min. For animal pretreatmentexperiments, mannoside ZFH-2056 was administered either IP (5 mg/kg) ororally (100 mg/kg) 30 min prior to inoculation with UTI189. Toaccurately count the number of IBCs, mice were sacrificed 6 hpi andbladders were aseptically removed, bisected, splayed on silicone platesand fixed in 2% paraformaldehyde (v/v). IBCs, readily discernable aspunctate violet spots, were quantified by LacZ staining of wholebladders.

Pharamacokinetic Analysis.

For intraperitoneal dosing, 50 μl of a 2 mg/ml (5 mg/kg) or 4 mg/ml (10mg/kg) solution of ZFH-2056 in PBS was injected into the peritonealcavity of the mouse. For oral dosing, 100 μl of a 20 mg/ml (100 mg/kg)solution of ZFH-2056 in 8% DMSO was inoculated with a gavage needle intothe mouse stomach. Urine was collected at 30 min, 1, 2, 3, 4, 6, and 8 hpost-treatment. An equal volume of 10 μM internal standard (ZFH-2050)was added to the urine. Mannosides were extracted from the urine byloading on C18 columns (100 mg, Waters), washing with 30% methanol, andeluting with 60% methanol. Vacuum-concentrated eluates were analyzedusing liquid chromatography-mass spectrometry system 30 with a lowerheated capillary temperature of 190° C. and a gradient as follows:Solvent B (80% acetonitrile in 0.1% formic acid) was held constant at 5%for 5 minutes, increased to 44% B by 45 minutes, and then to a 95% B by65 minutes. SRM mode quantification was performed with collision gasenergy of 30% for the following MS/MS transitions (precursor m/z/productm/z): compound ZFH-2056, 447/285; compound ZFH-2050, 390/228. Absolutequantification was achieved by comparison to a calibration curve.

Bladder Tissue Bacterial Titer Determination.

Mannoside ZFH-2056 was administered either IP (5 mg/kg) or orally (100mg/kg) 30 min prior to inoculation with UTI89. To enumerate the bacteriapresent, mice were sacrificed at 6 hpi and bladders were asepticallyremoved and homogenized in 1 ml PBS, serially diluted and plated onto LBagar plates. CFU was enumerated after 16 h of growth at 37° C.

Confocal Microscopy.

Mannoside ZFH-2056 was administered IP (5 mg/kg) 30 min prior toinoculation with UTI89. To visualize bacterial behavior within thebladder, mice were sacrificed at 6 hpi and bladders were asepticallyremoved, bisected, splayed on silicone plates revealing the luminalsurface and fixed in 2% paraformaldehyde (v/v). The splayed bladderswere then incubated for 20 min at room temperature with (i) SYTO9(1:1000 in PBS; Molecular Probes) to stain bacteria and (ii) Alexa Fluor594-conjugated wheat germ agglutinin (WGA; 1:1000 in PBS; MolecularProbes) to stain the bladder luminal surface. Bladders were rinsed withPBS, mounted using Prolong Gold antifade reagent (Invitrogen) andexamined with a Zeiss LSM510 confocal laser scanning microscope under a63× objective. SYTO9 and WGA were excited at 488 and 594 nm,respectively.

Gentamicin Protection Assay.

To enumerate bacteria present in the intracellular versus extracellularcompartments, bladders were aseptically harvested at 6 hpi. The bladderswere then bisected twice and washed three times in 500 μl of PBS each.The wash fractions were pooled, lightly spun at 500 rpm for 5 min topellet exfoliated bladder cells, serially diluted, and plated onto LBagar to obtain the luminal fraction. The bladders were treated with 100μg of gentamicin/ml for 90 min at 37° C. After treatment, the bladderswere washed twice with PBS to eliminate residual gentamicin, homogenizedin 1 ml of PBS, serially diluted, and plated onto LB agar to enumberatethe CFUs in the intracellular fraction.

Antibiotic Treatment.

Mice were given TMP-SMZ in the drinking water at a concentration of 54μg/ml and 270 μg/ml, respectively. Water was changed daily for 3 daysprior to inoculation with UTI89. Mice remained on TMP-SMZ during theinfection. To determine TMP-SMZ concentration in the urine, urine wascollected after 3 days of TMP-SMZ treatment and quantified by LC-MSfollowing addition of sulfisoxazole as an internal standard.

Growth Curve.

An overnight culture of PBC-1 was diluted 1:1000 in LB in the absence orpresence of TMP-SMZ and/or mannoside ZFH-2056. The highest concentrationof TMP-SMZ used was 512 μg/ml and 2560 μg/ml, respectively. Two-folddilutions of TMP-SMZ were performed. Mannoside ZFH-2056 was added at 100μM. Growth curves were performed in a 96-well plate at 37° C. with A600readings taken every 30 min for 8 h.

Hemagglutination Assay.

PBC-1 was grown statically in LB in the absence or presence of TMP-SMZfor 2×24 h at 37° C. The highest concentration of TMP-SMZ used was 256g/ml and 1280 g/ml, respectively. Two-fold dilutions of TMP-SMZ wereperformed. Hemagglutination assays for mannose-sensitive agglutinationof guinea pig red blood cells were performed as previously described.

Statistical Analysis.

Observed differences in bacterial titers and IBC numbers were analyzedfor significance using the nonparametric Mann-Whitney U test (Prizm;GraphPad Software).

Example 15. Improving Solubility and Potency of Mannosides

Amide groups were added to the insoluble 1ZFH253 and 1ZFH296 mannosidesto produce mannoside compounds with increased solubility as shown in theFIG. 24. A divalent compound was also produced using 2ZFH50. Theefficacy of these compounds along with 1ZFH177 was tested in an in vitroinhibition of hemagglutination and biofilm assays (FIG. 25). Theseresults demonstrate improved in vitro potency of mannosides comprisingenhanced solubility.

ZFH56 was injected IP into mice at various doses and levels of thecompound were measured in the animal (FIG. 26). 10 mg/kg, 5 mg/kg or 5mg/kg followed by a 4 hour boost were injected into animals.

IBC numbers were also determined in animals pretreated with ZFH56 (FIG.27). ZFH56 also altered the pathogenic pathway as measured by confocalmicroscopy (FIG. 28). ZFH56 also showed increased efficacy againstTMP-SMZ resistant strains (FIG. 29).

Example 16. Testing Against Various Inoculum Levels

An inoculum of 10⁷ is likely well above the physiological dose typicallyfound in a human. It is likely that only a few bacteria are inoculatedinto the urinary tract for a successful infection. Thus, a lower dose ofbacteria was introduced to the mouse and determined whether thisincreased the efficacy of mannoside ZFH56 (FIG. 30). In order to obtaina consistent infection in the mouse model of UTI the dose could only belowered by 10 fold. With an inoculum of UTI89 of 10⁶, there was asignificant decrease (p<0.0001) in bacterial titers in the ZFH56 treatedmice compared to the untreated mice. However, the level of infection inthe mannoside treated mice at both the 10⁷ and 10⁶ inoculum wasapproximately equivalent suggesting that the lower dose did not furtherenhance efficacy of the mannoside. Using IBC counts in the bladder atthe lower dose there were no IBCs observed in the ZFH56 treated mice atthe 10⁶ inoculum. However the overall number of IBCs in the untreatedmice were also much lower. Since there were no IBCs at 6 h in the 10⁶inoculum, it was hypothesized that the level of infection at 2 weekspost-infection would be significantly lower. Evaluation of 2 week titersrevealed a significant decrease in bacterial load in the mannosidetreated bladders with the 10⁶ inoculum. This data suggests that uponlowering the inoculum, mannoside is able to significantly reducecolonization at 2 wpi with a single dose of 5 mg/kg ZFH56 30 min priorto inoculation of UTI89.

PK studies with IP dosing revealed that after injection, mannoside ZFH56is rapidly eliminated in the urine and is mostly gone by 6-8 h postinjection. Thus the mice were boosted with 5 mg/kg mannoside ZFH56 every8 h to maintain a constant presence of mannoside in the mouse. Thisexperiment comprised 4 groups: (1) untreated, (2) pretreat by IPinjection of 5 mg/kg ZFH56 30 min prior to inoculation with UTI89, (3)pretreat by IP injection of 5 mg/kg ZFH56 30 min prior to inoculationplus 2 additional treatments every 8 h, and (4) inoculation with UTI89followed by IP treatment with 5 mg/kg ZFH56 1 h post-infection and 2additional treatments every 8 h. Mice were harvested at 24 hpi. This wasdone with the 10′ and 10⁶ inoculum of UTI89. There was a significantdrop in bacterial titers with the single pretreatment of ZFH56 with bothinoculum doses. In the mice inoculated with 10⁷ UTI89 there was furtherefficacy of ZFH56 upon boosting. However, this same effect was notobserved with the 10₆ inoculum. With both inoculums there was noefficacy observed upon post-infection treatment with ZFH56. These datasuggest that mannoside is currently most efficacious when usedprophylactically (FIG. 31).

Next, it was determined if a 10 fold increase in ZFH56 would increaseefficacy and if the divalent ZFH308 was as efficacious as ZFH56 at 5mg/kg (FIG. 32). Mice were treated with 5 mg/kg ZFH56, 50 mg/kg ZFH56 or5 mg/kg ZFH308 30 min prior to infection with 10⁷ UTI89. At 6 and 24 hpibladders were harvested and titered. At 6 hpi, 5 mg/kg ZFH56 and 50mg/kg ZFH56 both significantly inhibited bacterial infection. However,50 mg/kg ZFH56 did not significantly enhance efficacy over the 5 mg/kgZFH56 dose. 5 mg/kg ZFH308 did not significantly reduce bacterialinfection at 6 hpi. At 24 hpi, both 5 mg/kg and 50 mg/kg ZFH56significantly reduced bacterial titers. 50 mg/kg ZFH56 showed slightenhancement over 5 mg/kg ZFH56 relative to untreated mice. Again ZFH308did not show efficacy in reducing infection. Thus, 5 mg/kg ZFH56 seemsto be the optimal dose for inhibiting bacterial infection and thedivalent ZFH308 does not show enhanced efficacy over the monovalentcompound.

Example 17. ZFH56 Disrupts Preformed Biofilm

UTI89 was grown in LB in PVC plates and allowed to form biofilm at RTfor 24 or 48 h. At 24 or 48 h, varying concentrations of mannoside ZFH56was added. Biofilm was then allowed to grow for another 24 h at RT.Biofilm formation was then quantitated using the standard crystal violetassay. Results showed that ZFH56 was able to disrupt preformed biofilm(FIG. 32). At 24 h the IC50 for biofilm disruption was ˜6 μM and at 48 hthe IC50 was 25 μM. This shows that not only can mannoside inhibitbiofilm formation it can dissolve biofilm that has already formed.

Introduction for Examples 18-22.

Catheter-associated urinary tract infections (CAUTIs) often arise frommultidrug resistant Gram-positive and Gram-negative bacterialcolonization and biofilm aggregation on the surface of indwellingurologic devices such as urinary catheters, rendering treatment verydifficult. Uropathogenic Escherichia coli (UPEC), the primary cause ofcommunity-acquired UTI, account for 50% of nosocomial UTIs, includingCAUTIs. Yet, very little is known about its pathogenesis followingurinary catheterization, which results in the disruption of the normalmechanical and antimicrobial defenses of the bladder. Previous reportsusing human biopsies and rodent models of infections have shown that thecatheterized bladder is edematous and highly inflamed with immune cellinfiltration and pro-inflammatory cytokine production, an environmentquite different from that which UPEC encounters in a non-catheterizedbladder. We hypothesized that these profound catheter-related changesmay affect UPEC pathogenesis.

The UPEC pathogenic cascade has been extensively characterized in anon-catheterized murine model of cystitis. UPEC elaborate on theirsurface adhesive type 1 pili which mediate binding to and invasion ofsuperficial umbrella cells lining the bladder epithelium. Onceintracellular UPEC can escape into the cytoplasm, replicate rapidly andundergo morphological differentiation within bladder epithelial cells toproduce mature intracellular bacterial communities (IBCs) of ˜10⁴-10⁵bacteria with biofilm-like properties. UPEC then flux out from infectedcells and can reinvade neighboring cells and start the process de novo.This acute phase of UPEC infection can lead to the development ofchronic cystitis, pyelonephritis, and the formation of quiescentintracellular reservoirs (QIRs) with absence of bacteriuria. Detailedunderstanding of the critical steps of this pathogenic cascade has ledto the development of small molecule inhibitors called mannosides.Mannosides specifically target the bacterial type 1 pili tip adhesin,FimH, which binds to mannosylated residues present on the surface of thebladder epithelium. Rationally designed to interfere and prevent FimHinteraction with these residues, mannosides inhibit UPEC binding andinvasion of the superficial umbrella cells during urinary tractinfections (UTIs). Mannosides, in combination with existingantibiotic-based UTI therapy, have recently been shown to be effectivein preventing and treating UPEC infections in non-catheterized infectionmodels. The present study investigates whether this therapeutic approachcould be beneficial in the prevention and treatment of CAUTIs.

In Examples 18-22, the optimized murine model of foreign body-associatedUTI that closely mimics CAUTI was used to investigate the consequencesof urinary catheterization on the pathophysiology of UPEC infection. Forthese studies, several UPEC virulence parameters, including thecontribution of type 1 pili, IBC formation, and QIR reactivation, wereassessed. The results obtained indicate that urinary catheterizationprovides UPEC with the opportunity to exploit the extracellular milieuof the bladder via type 1 pili-mediated biofilm formation on the surfaceof the foreign body, which results in a shift in the niche population.Administration of mannosides in combination withtrimethoprim/sulfamethoxazole prior to urinary catheterization preventsUPEC colonization of the urinary tract. These Examples provide importantinsights into the mechanisms underlying UPEC-mediated CAUTI, and informsefforts to design better therapeutic approaches to prevent andpotentially treat these infections.

Materials and Methods for Examples 18-22

Bacterial Strains and Growth Conditions.

All strains used in this study and their characteristics are listed inTable 16. Unless otherwise specified, a single colony of E. coli grownon Luria Bertani (LB, Becton Dickinson) agar plate supplemented withappropriate antibiotics was inoculated into LB broth and grownstatically at 37° C. for 18 h.

TABLE 16 Strains used in this study Species and Relevant AntibioticStrains resistance Characteristics UTI89 Parental UPEC UTI89 strain,cystitis isolate UTI89ΔfimH UTI89 with an in- frame deletion of fimH,type 1 pili defective UTI89ΔsfaA-H UTI89 with an in- frame deletion ofsfa operon, S pili defective UTI89ΔsfaA- Kan^(R) Cm^(R) UTI89 within-frame HΔfimB-H deletion of the sfa operon and the fim operon fromfimB to fimH, S and type 1 pili defective UTI89ΔcsgA UTI89 with an in-frame deletion of csgA, curli deficient UTI89ΔcsgBΔcsgG UTI89 within-frame deletions of csgB and csgG, curli deficient UTI89HK::GFPKan^(R) UTI89 with an insertion of kanamycin cassette and GFP at the HKsite UTI89pCOMGFP Kan^(R) UTI89 ectopically expressing GFP from pCOMplasmidIn Vitro Cultivation and Quantification of Biofilms.

Biofilms were grown as described by Ferrieres et al. (2007; FEMS Immun.Med. Micro 51:212) on All Silicone Foley catheters (Bard Medical, GA) orsilicone tubing (Thermo Fisher Scientific Inc., PA) and modified asfollows. All tubing and connectors in the system were autoclaved andethanol sterilized prior to use. The system was assembled similar to thepreviously described flow-chamber system. Priming of the catheter or thesilicone tubing occurred at 37° C. for 20 min by flowing pre-warmedpooled human urine. Urine was collected from healthy volunteers asapproved by the Institutional Review Board of Washington University inSt. Louis. Pooled samples were spun at 10000×g for 15 min, filteredthrough 33 μM filters, and, if necessary, stored at 4° C. for no morethan 3 days. Three milliliters of stationary-phase E. colifrom overnightcultures were diluted to 1-2×10⁶ CFU/ml in human urine and injected intothe catheter or silicone tubing using a 30 cc gauge needle. The bacteriawere allowed to attach to the substratum for 1 h before urine flow viaWatson-Marlow peristaltic pump 205S was resumed at 0.5 ml min⁻¹. Whenindicated, urine was supplemented with 1% methyl mannose (Sigma, MO)prior to the experiment. After 24 hours, the remaining medium wasexchanged for sterile ddH₂O that was allowed to flow at 0.5 ml min⁻¹ toremove residual urine and non-adherent bacteria in the system. Theliquid from catheter or silicone tubing was then removed by capillaryaction onto absorbent paper. The tubing was cut into pieces for CFUenumeration or crystal violet staining, respectively. For CFUenumeration, at least three pieces (1 cm in length) of incubated tubingwere separately further cut into smaller pieces and placed into 1 mlPBS. Adherent cells were detached by sonication (10 min) and vigorousvortexing (3 min). Viable bacterial counts were assessed by serialdilution on LB plate with appropriate antibiotics. Crystal violetstaining was used to determine biofilm biomass. At least 3 pieces ofincubated tubing (3 cm in length) were filled with 0.5% crystal violetat room temperature for 10 min. Excess dye was removed by washing threetimes with ddH₂O and dried by capillary action on absorbent paper. Thebound crystal violet was then dissolved in 200 μl of 33% acetic acid andabsorbance measured at 595 nm. The amount of biofilm was expressed asCFU/ml per cm² and A595/cm². The experiment was repeated at least threetimes with different urine samples.

Animal Implantation and Infections.

Six to seven week-old female wild-type C57BL/6Ncr mice purchased fromthe National Cancer Institute (NCI) were used in this study. Experimentswere performed following one week adaptation in the animal facilityafter arrival from NCI. Animals were implanted and infected with theindicated bacterial strain as previously described. Briefly, seven toeight week-old female mice were anesthetized by inhalation of isofluraneand implanted with platinum-cured silicone tubing (4-5 mm in length)(Implanted). Immediately following implantation, 50 μl of ˜1-2×10⁷ CFUbacteria in 1×PBS were introduced in the bladder lumen by transurethralinoculation. Non-implanted animals were inoculated in the same manner.Animals were sacrificed at indicated time points by cervical dislocationunder anesthesia inhalation. Bladders and kidneys were asepticallyharvested. Subsequently, the silicone implant was retrieved from thebladder when present, placed in PBS, sonicated for 10 min and thenvortexed at maximum speed for 3 min. Bladder and kidneys from each mousewere homogenized in PBS. Samples were serially diluted and plated on LBagar plates supplemented with appropriate antibiotics. CFU wereenumerated after 24 h incubation at 37° C. In all cases, experimentswere performed at least twice with n=5 mice/strain/condition. Allstudies and procedures were approved by the Animal Studies Committee atWashington University School of Medicine.

Mannoside and Antibiotic Treatment.

SdfFor pretreatment experiments, 50 μl mannoside (mannoside 6; 5 mg/kgmouse body weight) or PBS was administered intraperitoneally 30 minprior to implantation as previously described. As indicated forpre-infection treatment, trimethoprim/sulfamethoxazole (TMP-SMZ) wasadded to the drinking water for three days prior to infection at 54 and270 μg/ml, respectively. The drinking water was changed every 24 h. Toassess the effects of mannoside and/or TMP-SMZ on establishedinfections, animals were implanted and infected for 24 h. At 24 hpi,TMP-SMZ was added to the drinking at the concentrations indicated aboveand mannoside 6 or PBS was administered i.p. 6 h prior to sacrifice.Animals were sacrificed 48 hpi.

UPEC Reservoir Reactivation.

Non-implanted animals were infected with UTI89HK::GFP as describedabove. At fourteen days post infection, urine was collected, seriallydiluted and plated for CFU and a subset of animals implanted asdescribed above. Animals determined to be bacteriuric (bacterial loadsgreater than or equal to 10⁴ CFU/ml in urine) as counted on titer platesthe next day were eliminated from further study. QIR reactivationpost-implantation was assessed by CFU enumeration of bacteria onimplants and in the organs 3 or 5 days post-implantation (17 or 19 dpi).UTI89HK::GFP titers greater than 10⁴ CFU/ml on implants or bladders wereconsidered reactivation events. Measures of reactivation events ofanimals, which were non-bacteruric at 14 dpi but non-implanted served ascontrols.

IBC enumeration and visualization. Implanted and non-implanted animalswere infected with UTI189 for 6 h. When indicated, mannoside 6 (5 mg/kg)or PBS was administered i.p. at 30 min prior to implantation. At 6 hpi,bladders were harvested, bisected, splayed on silicone plates and fixedin 2% paraformaldehyde. LacZ staining of whole bladders was performed aspreviously described. Punctate violet spots characteristic of IBCs wereenumerated by light microscopy.

For IBC visualization, animals were infected with UTI189 constitutivelyexpressing GFP (UTI89pCom-GFP). At the indicated time point, bladderswere removed, bisected, splayed, and fixed as described above. Thesplayed bladders were then incubated for 20 min at room temperature withAlexa Fluor 633-conjugated wheat germ agglutinin (WGA; 1:1000 in PBS;Molecular Probes) for staining of the bladder surface and, whenindicated, SYTO083 (1:1000 in PBS; Molecular Probes) to stain bacteria.Bladders were rinsed with PBS, mounted using Prolong Gold antifadereagent (Invitrogen) and examined with a Zeiss LSM510 confocal laserscanning microscope under a 63× objective. SYTO083 and WGA were excitedat 543 and 633 nm, respectively.

Gentamicin Protection Assay.

To quantify intracellular and extracellular bacteria, bladders wereaseptically harvested at 3 and 6 hpi. Bladders were cut in 4 parts andwashed three times in 500 μl PBS. The wash fractions were pooled,centrifuged at 500 rpm for 5 min to pellet exfoliated bladder cells. Thesupernatants were then serially diluted and plated on LB agarsupplemented with appropriate antibiotics, which were incubated at 37°C. for 24 hrs to obtain extracellular bacterial CFU counts. Rinsedbladders were then treated with 100 μg/ml gentamicin for 90 min at 37°C. Following gentamicin treatment, the bladder tissue was washed twicewith PBS to eliminate residual antibiotics, homogenized in 1 ml PBS, andbacterial CFU counts of determined as above to determine the levels ofintracellular bacteria (protected from gentamicin killing).

Statistical Methods.

Comparisons between groups were conducted by nonparametric Mann-WhitneyU test using GraphPad Prism (GraphPad software, version 5). Values belowthe limit of detection for in vivo experiments (20 CFU for organs, 40CFU for implants) were assigned the appropriate LOD value forstatistical analyses. All tests were two tailed, and a p-value less than0.05 was considered significant. Colonization and infection was definedas organs/implants with bacterial titers above the limit of detection.

Example 18: UPEC Adherence, Invasion, and IBC Morphology are Unalteredin Catheterized Bladders

IBC formation occurs in the pathogenesis of UPEC in non-catheterizedpatients and has been shown in mouse models to be critical forinfection. To assess the effects of urinary catheterization on IBCformation, 4-5 mm platinum-cured silicone tubing sections were implantedin the bladders of C57Bl/6Ncr female mice, which were then immediatelyinfected with 1-2×10⁷ CFU of the well-studied virulent UPEC strain UTI89by transurethral catheterization. Gentamicin protection assays performedat 3 hpi revealed no statistical difference in either the extracellularor intracellular UPEC populations in the presence or absence of implants(FIG. 33), indicating no gross defect in bacterial invasion in implantedanimals. IBC formation within both implanted and non-implanted bladderswas assessed by LacZ staining and confocal scanning laser microscopy(CSLM) at 6 hpi (FIG. 34A). Inoculation of UPEC into implanted animalsresulted in significantly fewer IBCs with a median of 8 IBCs/bladder(p=0.0044; FIG. 34B) compared to non-implanted animals in which IBCnumbers ranged up to >250 IBCs/bladder with a median of 55 IBCs/bladder.However, bacterial CFU in implanted bladders were similar to those innon-implanted animals (data not shown). To account for this observation,we postulated that IBC morphology might be different in implantedanimals. However, IBCs formed in implanted animals were observed to beoverall similar in size and shape as those produced in non-implantedbladders (FIG. 34C). UPEC were also seen to produce multiple IBCs withinone umbrella cell and filamentous bacterial clumps in both catheterimplanted and non-implanted bladders at 6 hpi (FIG. 35). UPECcolonization at 6 hpi was selectively localized in the remainingumbrella cells and not observed in the exposed underlying epithelium inimplanted bladders (FIG. 35). Together, these findings indicate thaturinary catheterization negatively impacts IBC formation by UPEC,possibly due to a correlated increase in exfoliation.

Example 19: Bacteria Originating from Existing UPEC Reservoirs can SeedUrinary Implant Colonization

One troubling possible outcome of the UPEC pathogenic cascade is theestablishment of quiescent intracellular reservoirs (QIR) in theunderlying epithelial layers, which have been shown can be a source ofrecurrent UTIs (rUTIs). QIRs were shown to be reactivated followingtreatment with protamine sulfate, a chemical that leads to exfoliationof the superficial umbrella cells of the uroepithelium. Like protaminesulfate, we have previously shown that urinary catheterization causessevere damage to the protective uroepithelial layer. Thus, wehypothesized that urinary catheterization might also reactivate existingUPEC reservoirs, resulting in bacteruria, catheter colonization andfurther dissemination. To test this hypothesis, mice were infected with1-2×10⁷ CFU UTI89HK::GFP and infection allowed to resolve over thecourse of 2 weeks. On day 14 post-infection, urine was collected fromeach animal to assess infection state prior to urinary implantation of asubset of these animals. Those animals in which titering of urines 14dpi indicating bacteriuria (≥10⁴ CFU/ml) were considered to have active(non-resolved and or recurrent) infection and were removed from furtheranalysis. The remaining mice were presumed to either have completelycleared the infection or to have established QIRs with bacteria loadsbetween 10¹ and 10⁴ CFU/ml. 3 or 5 days post implantation, reservoirreactivation was assessed by bacterial colonization of implants andbladder. On day 3 post implantation, UPEC UTI89HK::GFP was recoveredfrom implants in 3 of 26 implanted mice (˜11.5%). One mouse with implanthad bladder colonization greater than 10⁴ CFU/ml compared to none of the22 similarly infected but non-implanted animals (FIG. 36A). There was nosignificant difference between groups at 3 dpi. For mice assessed at 5days post-implantation, UTI89HK::GFP was recovered from implants of 4out of 32 animals (13%) with two of them having bladder titers greaterthan 10⁴ CFU/ml (FIG. 36B) compared to 0 out of 23 in non-implantedanimals. Interestingly, there were overall significantly fewer bacteriarecovered from the bladders of implanted animals compared tonon-implanted animals at 5 dpi (p=0.0010), suggesting that eitherreactivated reservoirs are cleared by the immune response prior to day 5following implantation or increased exfoliation prevents establishmentof persistent infections. Together, these data indicate that urinarycatheter colonization can occur from previous urinary infections even ifthose infections appear by bacteriuria counts to have been resolved.

Example 20: FimH is Required for Biofilm Formation and UPEC Colonizationof the Urinary Tract Following Catheter Implantation

Biofilm formation is a critical component of CAUTI pathophysiology. Wehave previously shown that UPEC is able to produce biofilms on thesurface of the foreign body and is recovered for the catheterized murinebladder at very high titers. Type 1 pili are major UPEC virulencefactors that have been shown to be critical for biofilm aggregation, IBCformation, and other aspects of UPEC uropathogenesis. Thus, we assessedthe contribution of these extracellular pili as well as other UPECfibers, including curli which contribute to biofilm formation or S piliassociated with E. coli clinical isolates producing strong biofilms, tobiofilm formation in filtered human urine under flow conditions andUPEC-mediated CAUTI in vivo. Deletion of the gene for the tip adhesin oftype 1 pili, FimH, in UTI89 resulted in significantly (p<0.0001) lowerbiomass (FIG. 37A) and an approximate 2-fold reduction in adherentviable bacteria (FIG. 37B) in biofilms formed in human urine in vitro.These data indicate that type 1 pili are a major contributor to UPECbiofilm formation in urine. The biofilm defect was specificallyassociated with the fimH mutant under these conditions, and was notobserved following deletions of the sfa operon to prevent S piliformation, csgA required for curli fiber formation, or a component ofthe flagellar system fliC (data not shown).

In vivo, similar to findings in a murine model of cystitis, UTI89ΔfimHis severely attenuated in the murine model of foreign body-associatedUTI (FIG. 37C). UTI89ΔfimH displayed >3 log fewer CFU in the bladder andwas unable to ascend to the kidneys at 24 hpi. Further, deletion of fimHresulted in significant reduction in implant colonization (p<0.0001).Similar to in vitro experiments, S-pili are not required for CAUTI sinceUTI89ΔsfaA-H is as virulent as wildtype UTI89 and a double deletion ofboth sfaA-H and fimB-H recapitulated the UTI89ΔfimH phenotype (FIG.38A). Furthermore, components of the curli system important for biofilmformation in vitro under certain conditions, but not in human urine(FIG. 37A-B), were also dispensable during CAUTI (FIG. 38B). Theresidual binding to implants and bladders in implanted animals couldtherefore be attributed to other pili or biofilm determinants. Together,these data strongly suggest that the tip adhesin FimH of type I pili isa critical determinant of UPEC virulence in mediating biofilm formationand virulence during CAUTI.

Example 21: Mannoside Treatment Reduces IBC Formation

Having established that FimH is required for UPEC virulence in implantedbladders, we investigated this as a potential therapeutic target forCAUTI using small molecules inhibitors designed to interfere with FimHbinding to mannosylated residues. This family of small molecules, calledmannosides, has recently been shown to prevent acute and chronic UPECinfections and potentiated the effectiveness of antibiotics incombinatorial treatment.

To investigate the potential therapeutic effects of mannosides on CAUTI,we first assessed the inhibitory effects of methyl-α-D-mannopyranoside(methyl mannose), on UTI89 biofilm formation in urine under flow.Similar to the deletion of fimH (FIG. 37A), UTI89 biofilms grown inpresence of 1% methyl mannose had significantly reduced biomass(p=0.0022) and biofilm-adherent cells (p=0.0012), compared to untreatedcontrols (FIG. 39). Since methyl mannose is a FimH antagonist, thesedata confirm the critical role of type 1 pili to biofilm formation inurine as was previously described for biofilms formed in LB media.

The effects of mannoside treatment were then assessed in vivo by usingIBC formation as well as implant and urinary tract colonization asbenchmarks of disease progression. Mice were treated intraperitoneally(i.p.) with saline or 5 mg/kg of mannoside 6, which is more potent thanmethyl mannose in vitro and in vivo, in PBS 30 min prior to urinaryimplantation. Catheter implantation was immediately followed bytransurethral inoculation of UTI89. IBC formation and bacterialcolonization were assayed by LacZ staining and CFU enumeration ofimplants, bladders, and kidneys at 6 hpi and 24 hpi, respectively. Asshown in FIGS. 40A and 40B, mannoside treatment further reduced IBCformation (p=0.0051) and bladder colonization (p=0.0114) in implantedanimals at 6 hpi, suggesting that this treatment prevents intracellularinfection. While eliminated from their intracellular niche, data furtherindicated that UPEC were able to persist in the extracellular milieuwhere they can colonize the surface of the implants to relativelysimilar levels as saline-treated animals (p=0.0547) (FIG. 40B). Nostatistical difference was observed in kidney colonization in thepresence or absence of mannosides (FIG. 40B). By 24 hpi, a time point atwhich the mannosides have been eliminated from the bladder, similarbacterial loads were recovered from implants, bladders, and kidneys inimplanted animals in the presence or absence of mannoside treatment(data not shown).

Example 22: Mannoside Treatment Increases the Efficiency of TMP-SMZ inPreventing UPEC Colonization

In order to examine whether mannosides could prevent establishment ofCAUTI when used in combination with antibiotics, animals were treatedwith 54 and 270 μg/ml of TMP-SMZ, respectively, in their drinking waterfor three days and then treated with saline or mannoside (5 mg/kg) i.p.30 min prior to implantation and bacterial inoculation. At 6 hpi, UPECcolonized the implants and bladders at significantly lower levels inanimals that only received antibiotics compared to those who receivedwater or were only administered mannoside (FIG. 40B). Interestingly,mannoside treatment in addition to TMP-SMZ further decreased UPECcolonization of implants, bladders, and kidneys compared to treatmentwith antibiotic alone (p<0.0005 in all cases). Furthermore, treatmentwith mannosides alone did not reduce bacterial titers from a 24 h oldUPEC infection and in combination with TMP-SMZ showed no additiveeffects on established UPEC CAUTI 24 hpi (data not shown). Together,these findings indicate that virulence-targeted therapies in combinationwith established antibiotic treatment can help prevent or delay theonset of CAUTI and that further research is warranted for enhancingmannosides potential as therapeutics against CAUTIs.

Discussion for Examples 18-22

UPEC is the major etiological agents of CAUTI. Yet, the molecularmechanisms of urinary catheter and bladder colonization followingurinary catheterization have not been elucidated. Studies in anoptimized murine model of foreign body-associated UTI10 show thaturinary catheterization favors UPEC exploitation of the bladderextracellular milieu. This occurs via type 1 pili-dependent biofilmformation on the surface of silicone implants in the murine bladder. Thedata further indicate that of the biofilm determinants tested, type 1pili are necessary for implant, bladder, and kidney colonization duringCAUTI; providing definitive experimental evidence for previous reportspostulating that type 1 pili may be required for UPEC persistence duringCAUTIs. Interestingly, fimH-deficient UPEC strains have the ability toadhere to some extent to the surface of the foreign body, probably usingother biofilm determinants such as other chaperone-usher pili systems,curli, or surface adhesins.

In addition to colonizing the foreign body in the bladder lumen, UPEC isable to exploit intracellular niches in implanted animals, albeit to alesser degree than in non-implanted animals, by invading and producingIBCs in the early stages of infection. Reduced IBC formation inimplanted animals may be a result of loss of the host superficial facetcells in which IBCs form due to increased exfoliation or damage to theuroepithelium following urinary catheterization. Nonetheless, thisfinding is of particular interest for treatment strategies againstUPEC-mediated CAUTI. In humans, removal of the contaminated urinarycatheter is the preferred method for treatment of these infections;however, the presence of bacteria in an intracellular compartmentprotected from host immune defenses and antibiotic treatment requiresmore comprehensive approaches as intracellular UPEC can lead tore-infection of a novel catheter or serve as a nidus for future UTI.

Quiescent intracellular reservoirs are an important outcome of UPECpathogenic cascade because they are proposed to be a mechanism ofrecurrent UTI following damage to the uroepithelium. Findings from thecurrent study indicate that urinary implantation of animals with ahistory of UTI can lead to bladder infection and implant colonizationwith the UPEC causing the first infection. This finding suggests that inaddition to introduction of extracellular and periurethral bacteria,urinary catheter colonization can occur from bacteria originating frompre-existing reservoirs or other niches within the urinary tract notappreciated by assessment of bacteriuria. Interestingly, by day 5following implantation in animals with a history of UTI, there isoverall a significant reduction in the number of QIRs compared to thatin non-implanted animals as assessed by bladder CFU. This reduction inbacterial load could be a result of enhanced immune-mediated clearanceof infected cells or to exfoliation of infected cells in implantedanimals. These hypotheses are currently being evaluated.

The identification of FimH as a critical virulence factor during UPECCAUTI provides an interesting avenue for the development of novelpreventative measures against these infections. In fact, there hasrecently been an upsurge in recommendations and guidelines formanagement of CAUTIs and rUTIs in catheterized patients, with aparticular focus on preventative measures including the limited use ofcatheters and even the prophylatic use of antibiotics prior andfollowing catheter removal. However, the inappropriate use ofantibiotics can worsen the problems of increasing antibiotic resistance.Accordingly, the present study suggests that the use of small moleculesinhibitors, such as mannosides, in combination with existing UTItreatment regimes, can lead to prevention or delay of UPEC colonization.Mannosides, were rationally designed to interfere with FimH-mediatedbinding to mannosylated proteins on the surface of the uroepithelium.Recent studies show that the use of mannosides in combination withantibiotics is highly effective in preventing IBC formation and acutestages of UPEC infection as well as treating chronic cystitis.Similarly, pretreatment with mannosides further prevents IBC formationfollowing UPEC infection of implanted bladders and reduces UPEC bindingto the uroepithelium. However, the presence of the abiotic implantsurface provided a favorable environment for adherence of extracellularbacteria. The inability of mannoside treatment to eliminate UPEC fromthe implant, given that the experimental evidence that mannose inhibitsUPEC biofilm on catheter material in urine in vitro, may be due to thelack of urine flow in this implanted murine model. It is possible thatif it were possible to truly catheterize mice in a manner analogous toclinical catheterization of humans in which urine flows through thecatheter that mannoside may have a more efficacious effect on implantclearance. Nonetheless, by preventing invasion and shifting UPEC's nicheto the extracellular milieu, mannosides enhanced the bacteriocidalefficacy of antibiotics, such as TMP-SMZ, which does not cross host cellmembranes. Mannosides could also be used in combination with bacterialinterference strategy being offered as alternatives in the prevention ofCAUTI. Previous reports have shown that pre-colonization of urinarycatheters with an avirulent E. coli strain 83972 delays the onset ofCAUTI in catheterized patients. Thus, it is quite possible that if usedin combination with mannosides, UPEC will be kept from the intracellularniche as well as from binding to the catheter during bacterialinterference with the avirulent strain. An attractive avirulent strainshould be able to colonize the catheter in a type 1-independent mannerto prevent invasion into urothelial cell, however E. coli strain 83972enhanced binding to silicone catheter requires ectopic expression offim. However, the therapeutic effects of mannoside 6 could not berecapitulated in our model of CAUTI (data not shown), possibly becausethe small molecules are ineffective at disrupting established biofilmsin vivo or that UPEC may employ additional type 1 pili-independentmechanisms for maintaining biofilms. It is a well-established fact thatthe extracellular matrix of bacterial biofilms is impermeable toantimicrobial and antibiotics, providing a safe haven for the microbeswithin. Further research is thus needed for better therapeutics againstCAUTIs. Nonetheless, the prophylatic use of mannosides prior to urinarycatheterization can help reduce the rate of recurrent UTIs fromintracellular bacterial reservoirs following the removal of contaminatedcatheters.

Urinary catheterization is a necessary medical procedure that causesmajor damage to the urinary tract. Pathogens, such as UPEC, takeadvantage of this compromised environment to exploit new and existingniches and establish severe infections. This report uncovers importantmolecular mechanisms underlying UPEC pathogenesis following urinarycatheterization. It raises important questions regarding the deleteriousconsequences of urinary catheterization and the origins of urinarycatheter colonization. These novel aspects of CAUTI pathophysiology,especially the presence of intracellular bacterial niches even inpresence of urinary catheters, should thus be taken into considerationfor better diagnosis and the development of anti-virulence basedpreventative and therapeutic approaches against these infections.

Example 23. Treatment of Chronic Mice with 5 Doses of Mannoside 8Eliminates UPEC from the Bladder

Mice were infected with the uropathogenic E. coli (UPEC) strain UTI89.Animals that established chronic infection were treated at day 12 postinfection with 5 doses of 50 mg/kg of Mannoside 8 administered 8 h apart(mice treated with PBS in the same way were included as controls). Micewere sacrificed 48 h post initial dose and bladder titers wereenumerated.

The results clearly show a greatly reduced titer of UPEC in mice treatedwith Mannoside 8 versus control mice treated with PBS (FIG. 41).

Example 24. Mannoside 8 is Effective Against the Multidrug ResistantUPEC Isolate EC958

Mice were infected with the multidrug resistant (MDR) UPEC strain EC958.Animals that established chronic infection were treated at day 12 postinfection with 1 dose of 50 mg/kg of Mannoside 8 (mice treated with PBSin the same way were included as controls). Mice were sacrificed 6 hpost initial dose and bladder titers were enumerated.

The results show that titers of MDR UPEC in mice treated with Mannoside8 had significantly lower titers of the MDR UPEC compared to MDR UPECtiters in control mice treated with PBS (FIG. 42).

Introduction for Examples 25-29

The development of antibiotics to broadly treat infections has led tosignificant advances in human health. Increasing bacterial resistance totraditional antibiotics and the dearth of programs to develop innovativeantibiotics threatens to reverse these advances. This has been describedas an impending “public health crisis”. For example, over 15 millionwomen suffer from urinary tract infections (UTI) annually in the U.S.with an estimated cost exceeding $2.5 billion. Uropathogenic E. coli(UPEC) account for up to 85% of all UTIs, which are exacerbated byincreasing antimicrobial resistance. Resistance of UPEC to theantibiotic cocktail, trimethoprimsulfamethoxazole (TMP-SMZ) hasincreased significantly in the past decade and thus therapy hasincreasingly required the use of last-line antibiotics such asfluoroquinolones, leading to increased treatment costs and an associatedrise in multidrug resistance. These developments make UTI one of themost visible manifestations of increasing gram-negative antibioticresistance. Recurrent UTI in healthy women is a major problem despitethe fact that standard antibiotic treatment typically results inclearance of bacteriuria.

The two relevant niches during acute UTI are tissue and urine. Theability of bacteria to associate within the tissue niche is critical forestablishing infection. Extracellular adhesive fibers known as pili arepromising targets of anti-virulence therapeutics as they are criticalfactors for colonizing and invading host tissues and forming biofilms.UPEC use the chaperone/usher pathway to assemble type 1 pili tipped withthe FimH adhesin. FimH mediates binding to mannosylated receptorspresent on the luminal surface of mammalian bladder epithelial cells,which facilitates bacterial invasion into these cells. Subversion ofinnate expulsion mechanisms requires entry into the cytoplasm where asingle UPEC can replicate rapidly into 10⁴-10⁵ bacteria, which aggregatein a type 1 dependent manner into a biofilm-like intracellular bacterialcommunity (IBC) that protects UPEC from host defenses and antibiotics.Bacteria later disperse from the IBC to spread to neighboring cells.FimH-dependent IBCs and the filaments that emerge from them commonlyexist in women with recurrent cystitis as revealed in a clinical studyof 100 women as IBC formation is thought to represent a mechanism thatallows UPEC to rapidly expand in numbers. Virulence factors thatincrease the fitness of UPEC in the urinary tract are predicted to beunder positive selection and the fimH gene is under positive selectionin clinical isolates of UPEC, consistent with its role in human disease.FimH is found in virtually all UPEC, the mannose binding pocket isinvariant and mutations which disrupt mannose binding are attenuated.Previous studies support the utility of mannosides, to disrupt FimHfunction, as an effective therapeutic strategy to treat UTI our refs.The rational X-ray structure based design and synthesis of novelbiphenyl mannosides FimH inhibitors were recently described, startingfrom phenyl mannoside 1 (FIG. 43A). In the following examples, it isshown that lead compound 6 (2ZFH56) and newly developed orallybioavailable mannosides are potent and fast acting antibacterialcompounds. It is demonstrated that these mannosides are effective attreating and preventing UTI in mice as well as potentiating theantimicrobial effects of TMP-SMZ.

Example 25. Mannosides Disrupt and Inhibit the Formation of Biofilms

Functional activity of mannoside FimH inhibitors 1-6 (FIG. 43A) wasfirst assessed in vitro using a hemagglutination and UPEC biofilmassays. The relative ability of compounds to block biofilm formation anddisrupt preformed biofilms was used to prioritize and select compoundsfor further in vivo evaluation. Biofilm formation is complex and themultiple determinants that contribute to their development andmaintenance may vary depending on growth conditions, medium andsubstrates. E. coli biofilm formation in LB at room temperature onpolyvinyl chloride (PVC) is dependent on type 1 pili and therefore theseconditions were used to determine the efficacy of the mannosides. Themedian inhibitory concentration (IC₅₀) values for compounds 1-3 and 6were all low micromolar but compound 6 showed the best activity with anIC₅₀ of 0.74 μM (FIG. 43B). In addition to preventing the formation ofbiofilms, it was also found that the mannosides inhibited the buildup upof preformed biofilms (FIG. 43C). Confocal microscopy of preformedbiofilms treated with mannoside 6 showed effective disruption of apreformed biofilm, likely explaining in part, the activity seen in FIG.43C. Furthermore, preformed biofilms treated with 6 lacked continuity asseen by gaping holes and lack of the tall mushroom-like structuresobserved in untreated biofilms (FIG. 43D, E). The propensity of E. colito form biofilms contributes to antibiotic treatment failures sinceantibiotics are unable to penetrate the dense biofilm matrix providingcompelling evidence that biofilm inhibitors can potentiate the effectsof antibiotics.

Example 26. Mannoside is Efficacious at Clearing Severe Infection

From the in vitro studies in Example 25, mannosides 4 and 6 showedsimilar activity in the hemagglutination inhibition (HAI) assay which ispredictive of relative activity in biofilm inhibition. However, theester group in compound 4 was unstable for oral dosing and it was foundthat its hydrolysis product 5 was 13-fold less potent. Mannoside 6 whichcontains an amide in place of the ester, was not only equipotent to 4but was also more stable plus had increased solubility and thus wasselected as our lead compound for initial in vivo evaluation.Pharmacokinetic (PK) studies with lead compound 6 in mice was performedusing intraperitoneal (IP) injection and oral (PO) gavage. Following IPdosing, 6 concentrations in the urine were quantified at several timepoints using HPLC and mass spectrometry (MS). Doses of 5 mg/kg and 10mg/kg resulted in concentrations of 1 mM in the urine 30 min aftertreatment (FIG. 44A). Eight hours after administration, 6 levelsremained near the IC₅₀ (0.74 μM) of biofilm inhibition. The mouse PK of6 dosed orally was next evaluated at several concentrations up to 200mg/kg. A 100 mg/kg dose of 6 resulted in 3-fold higher concentrationsrelative to IP (10 mg/kg) eight hours post-dosing demonstrating someoral bioavailability of 6. It is also noteworthy that >95% of drug wasexcreted in the urine unchanged and no apparent toxicity was observed upto a 200 mg/kg dose as measured by survival and weight gain/loss.

In order to determine the therapeutic potential of 6 for treatingchronic UTIs, a unique preclinical murine model was adopted. In humans,the ultimate outcome of UPEC infection of the urinary tract ranges fromasymptomatic bacteriuria to acute self-limiting infection tochronic/recurrent UTI. Similarly, the outcome of UTI in C3H/HeN miceranges from self-limiting to long-lasting, chronic cystitischaracterized by persistent, high titer bacteriuria (>10⁴ colony formingunits (CFU)/ml), high titer bacterial bladder burdens at sacrifice >2weeks post-infection (wpi), chronic inflammation, and urothelialnecrosis. The acute host response, within the first 24 hours, totissue-associated UPEC has been shown to determine disease outcomeincluding predisposition to chronic/recurrent UTI. Thus, C3H/HeN micewere infected with 1×10⁷ CFU of UTI89 and mice developing chroniccystitis, as determined by persistent urine titers of >10⁶ through 2weeks post infection (wpi), were PO treated at 2 wpi with 6 at a singledose of 100 or 50 mg/kg to evaluate the ability of mannoside to treatUTI. 6 hours post-treatment bacterial counts in the bladder wereenumerated. A dramatic 3-log drop in bacterial titers was observedsuggesting mannoside is efficacious at clearing severe infection within6 hours of oral delivery of a chronic long lasting infection (FIG. 44B).

Example 27. Prophylactic Use of Mannoside

Since 6 successfully treated chronic cystitis, it was of interest toelucidate if mannosides could also prevent a UTI as a prospectiveprophylactic therapy. To mimic this clinical scenario, the efficacy of 6in in vivo treatment was evaluated by dosing mice either IP or PO 30 minprior to infecting with UTI89. At 6 hours post infection (hpi), bladderswere removed and total bacterial CFUs were quantified. In both the IPand PO treated cohorts a significant drop in bacterial counts wasobserved, demonstrating the efficacy of 6 in reducing overall UPECcolonization of the bladder (FIG. 44E). Furthermore, there was also asignificant reduction of IBCs in the mice that were pretreated withmannoside (FIG. 44F). To demonstrate 6 reduced IBC formation by blockingUPEC invasion into the bladder tissue, gentamicin treatment assays wereperformed. Gentamicin kills extracellular UPEC but is unable topenetrate tissue and thus intracellular bacteria survive treatment. Itwas found that in the 6-treated mice, gentamicin treatment of thebladders eliminated all CFUs (FIG. 44G). In bladders from untreatedmice, 10³-10⁴ CFUs remained after gentamicin treatment, reflecting thebacteria present that had invaded the bladder epithelium and thuscircumvented the treatment. Confocal microscopy of bladders in theuntreated cohort showed normal, robust IBC formation (FIG. 44C) whereasIBCs were rarely seen in the mannoside treated mouse bladders howeverbacteria were observed in the bladder luminal compartment (FIG. 44D).These results demonstrate that a mannoside FimH inhibitor preventsbacterial invasion into the bladder tissue and significantly reducesinfection in the bladder. This novel class of orally active biphenylmannosides has potential utility for the treatment of women sufferingfrom chronic/recurrent UTIs as an alternative to prophylactic antibiotictreatment and would significantly benefit women with increased incidenceof UTI due to sexual intercourse.

Example 28. Mannosides Inhibit the Invasion of UPEC into the BladderTissue and Potentiate the Efficacy of TMP-SMZ

The first-line treatment of choice for UTI has traditionally been a3-day course of TMP-SMZ. Women suffering from chronic/recurrent UTIs areoften given TMP-SMZ prophylactically to prevent recurrence. However,resistance to this TMP-SMZ regimen is rapidly expanding. It washypothesized that by preventing bacterial invasion into the bladdertissue, a FimH inhibitor may result in anti-virulence synergism withTMP-SMZ and may curtail or circumvent the problem of TMP-SMZ resistance.This theory was evaluated in a preclinical animal model where mice givenTMP-SMZ for 3 days were infected with either UTI89 or the TMP-SMZRstrain, PBC-1. Mice were IP treated with 6 30 min prior to inoculationwith bacteria and compared to a control group of untreated animals.After inoculation with UTI89 or PBC-1, bacterial CFUs were quantified at6 hpi. As expected, treatment with TMP-SMZ alone resulted in asignificant drop in bacterial load in the UTI89-infected mice but had noeffect on PBC-1, since it is resistant to TMP-SMZ. Upon treatment with 6alone there was a significant drop in bacterial load of both strains inthe bladder. In the dual treatment group there was also a significantdrop in bacterial CFUs compared to mannoside alone or TMP-SMZ alone forboth strains which was most pronounced for PBC-1 (FIG. 45). It wasdetermined that the presence of mannoside had no effect on growth orkilling efficiency of either strain during growth in vitro in thepresence or absence of TMPSMZ. Therefore, the observation that incombination with 6, the TMP-SMZR strain PBC-1 succumbed to antibiotictreatment suggested that the mannoside potentiates the efficacy ofTMP-SMZ by a unique mechanism. Based on growth curves in TMP-SMZ, PBC-1was calculated to have a Minimum Inhibition Concentration (MIC) of 256and 1280 μg/ml for TMP and SMZ, respectively and UTI89 was calculated tohave an MIC of 0.05 μg/ml TMP and 0.25 μg/ml SMZ. The presence ofmannoside had no effect on growth or killing efficiency of eitherstrain. It is well established that TMP concentrates in the urine andthis serendipitous feature is a major reason TMP-SMZ has been thepreferred antibiotic for UTI over the last several decades. Usingquantitative HPLC-MS, the concentration of TMP-SMZ was measured in theurine of mice after 3 days of treatment with 54 μg/ml and 270 μg/ml TMPand SMZ, respectively. TMP concentrations were determined to be9.95+/−4.36 mg/ml and SMZ at 67.17+/−32.51 μg/ml. These results indicatethat by preventing bacterial invasion, 6 compartmentalizes the microbesto the bladder lumen thus exposing them to TMP-SMZ concentrations abovethe MIC of PBC-1, resulting in augmentation of bacterial cell killing.Presumably TMP-SMZ concentrations reach tissue concentrations above theMIC needed for UTI89 killing but fail to reach tissue levels needed forkilling PBC-1. These results clearly highlight the importance of theintracellular pathway in bacterial persistence. In addition to escapingthe immune system in their intracellular niche, bacteria are also ableto evade exposure to antibiotics as highlighted by the clinicallyTMP-SMZ resistant strain. In summary, mannosides could benefit thosewomen on suppressive antibiotic therapy by inhibiting the invasion ofUPEC into the bladder tissue and potentiating the efficacy of TMP-SMZcreating a cost-effective treatment, which is predicted to lower therate of treatment failures.

Example 29. Mannoside Optimization

While 6 shows good efficacy in vivo, it was sought to identify optimizedmannosides with further improved pharmacokinetics in particular thoseencompassing increased cell permeability and thus better oralbioavailability and bladder tissue penetration. The inherent polarity ofmannosides and other sugar-derived compounds often limits their cellularpermeability and increasing their hydrophobicity (Log P/Log D) ispredicted to improve the latter. Computational modeling of mannosidesbound to FimH suggested that the ortho-position of the biphenyl ringattached to mannose is aimed at Tyr137 and improved hydrophobic contactcould be achieved by substitution. The increased hydrophobicity was notonly predicted to improve FimH binding affinity but also the oralbioavailability and bladder tissue penetration relative to startingmannoside 6. Furthermore, these derivatives will likely displayincreased metabolic stability through protection of the glycosidic bondfrom hydrolysis both in the gut and by α-mannosidases. Thus, a matchedpair analysis was performed of monoamide 3 compared to ortho-substitutedanalogs bearing methyl, trifluoromethyl, and chloro groups. Thecompounds were evaluated for their potency in the hemagglutinationinhibition (HAI) assay and it was discovered that all biphenyl ringsubstitutions yielded more potent inhibitors (FIG. 46A). Ortho-chloromannoside 7 inhibited hemagglutination with a potency of 125 nM which is10-fold better than matched pair 3 while the ortho-methyl analog 8 waseven 2-fold more active (HAI=62 nM). Substitution with trifluoromethylgave the most potent analog 9 with an HAI=32 nM. This data suggests thatour prediction for increased hydrophobic contact with Tyr137 mightexplain this enhanced potency since the trifluoromethyl analog 9 has thelargest hydrophobic surface area and also shows the highest activity.However, it is also possible that the orientation of the phenyl ringsare altered slightly and conformationally restricted in a moreproductive form conducive to improved FimH binding. In any case, theoutcome of this preliminary study directed us to developortho-trifluoromethyl diamide 10 which is exponentially more potent thanany previously reported mannoside FimH inhibitor with an HAI=8 nM. Thisunprecedented level of potency corresponds to a 15,000-fold improvementover butyl-α-D-mannoside (125 μM) and is 50-fold better than lead 6.Based on their improved in vitro properties, we tested the optimizedmannosides for mouse oral PK (FIG. 46B) and found at 50 mg/kg compounds8 and 10 yielded the highest concentrations in urine at 6 hours postdosing with 8 displaying equivalent concentrations to 100 mg/kg ofmannoside 6. Due to their enhanced potency and optimal PK, mannosides 8and 10 were selected for further testing in our chronic infection mousemodel. C3H/HeN mice having chronic cystitis at 2 weeks post infectionwere treated with 6, 8 or 10. Treatment with 8 and 10 resulted in asignificant and dramatic 4 log reduction in bacterial counts in thebladder 6 hours posttreatment (FIG. 4c ). Although ortho-trifluoromethyldiamide 10 is 8-fold more potent in vitro than ortho-methyl monoamide 8,it was found that when dosed orally at 50 mg/kg 8 showed better efficacyin vivo. 8 reduced bacterial CFUs in the bladder almost 2 Log unitsbetter than 6 at the identical 50 mg/kg dose and was still moreefficacious than a 100 mg/kg dose of 6. These results can be explainedby increased FimH inhibition combined with improved PK of theortho-substituted mannosides relative to 6. Mannoside 8 represents avery promising lead preclinical candidate for the oral treatment andprevention of recurrent urinary tract infections.

Discussion for Examples 25-29.

The efficacy of anti-virulence compounds in vivo has not beenextensively characterized. Herein, the most potent orally active smallmolecule FimH antagonists described are reported to date. This is alsothe first demonstration that a mannoside FimH inhibitor showstherapeutic potential for treating an established chronic urinary tractinfection in vivo. These innovative compounds also show excellentefficacy in vivo when used prophylactically. Furthermore, the mechanismof action displayed by FimH inhibitors keeps bacteria extracellular andsensitizes a TMP-SMZ resistant strain by prolonging exposure toantibiotic levels above its MIC. This enhanced susceptibility may helpovercome the rising problem of TMP-SMZ resistance amongst E. coli. Notonly are these anti-virulence FimH antagonists effective as a treatmentagainst UTI, but their oral availability represents a major step towardeffective preclinical optimization and drug development. Alternativemanagement strategies are needed for patients suffering fromchronic/recurrent UTIs. Prophylactic administration of mannoside aloneor in combination with TMP-SMZ could potentially reduce the incidence oftreatment failure and shorten the time span of currently administeredsuppressive therapy. A shift to reduce the use of fluoroquinolones wouldprovide both a more cost-effective treatment option as well as slow thespread of resistance to this class of antibiotics. Given that resistanceto antibiotic therapy is rapidly increasing, it is time to reconsiderstandard UTI therapy in order to preserve the effectiveness of currentantibiotics. The use of FimH inhibitors as a targeted therapeuticstrategy benefits from the fact that it is not broad spectrum and willspecifically target those bacteria expressing type 1 pili, ubiquitousamongst UPEC. Given the unique welldefined clinical population ofpatients with UTI, of which nearly 85% of the cases are caused by UPEC,the narrow spectrum afforded by mannosides provides an advantage overalternative therapies and other broad spectrum anti-virulence drugscurrently in clinical trials. It is predicted that mannosides eitherdosed alone or in combination with commonly used antibiotics can treatalmost all initial and chronic UTIs.

Outside of UTI, cell-cell adherence to host tissues is the first step inmost infectious diseases. Thus, due to the commonality of adhesionmechanisms in bacterial and viral pathogenesis, similar but uniqueanti-virulence compounds can be tailored to treat a multitude ofinfectious diseases. The results in Examples 25-29 validate the utilityof employing a rational approach to study the molecular mechanisms ofpathogenesis and resulted in the discovery and development of potentialanti-virulence therapeutics, which specifically target mechanismsessential in UPEC pathogenesis. In conclusion, these studies have thepotential to revolutionize current approaches to both antimicrobial andanti-viral treatment and provides not only new opportunities foreffectively treating a broad range of infectious diseases, but also thepotential to curtail the ever expanding medical crisis of pathogenresistance to traditional antibiotics.

Methods for Example 25-29.

UTI189 biofilm was grown in LB+/−mannoside for 24 h at 22° C. in PVCplates and quantified using crystal violet. UTI189 biofilm for confocalmicroscopy was grown in LB for 24 h at 22° C. on PVC coverslips followedby mannoside treatment for 16 h. For all animal experiments UTI189 orPBC-1 was grown 2×24 h statically in LB at 37° C. and inoculated at adose of 1×107 bacteria in 50 μl. All mice used were female C3H/HeN(Harlan). For the chronic UTI model, mice were infected for 2 weeksprior to treatment with mannoside. For IP dosing, 50 μl of 2 mg/ml (5mg/kg) or 4 mg/ml (10 mg/kg) 6 in PBS was injected into the mouse 30 minprior to inoculation of bacteria. For oral dosing, 100 μl of 10 mg/ml(50 mg/kg) or 20 mg/ml (100 mg/kg) mannoside in 8% DMSO was inoculatedwith a gavage needle 30 min prior to inoculation of bacteria. Massspectrometry was used to quantify urinary mannoside or TMP-SMZconcentrations. For CFU counts, bladders were harvested at 6 hpi andplaced in 1 mL PBS. Bladders were then homogenized, diluted and platedon LB. After growth at 37° C. overnight, bacterial counts weredetermined. LacZ staining and gentamicin protection assays wereperformed at 6 hpi. For antibiotic experiments, mice were given TMP-SMZin the drinking water at a concentration of 54 μg/ml and 270 μg/ml,respectively. Water was changed daily with fresh antibiotics. Standardgrowth curve and hemagglutination assays were performed. All statisticalanalysis performed was a two-tailed Mann-Whitney U test. Compounds 1-6were prepared as outlined in Han et. al. (2010; J. Med Chem. 53:4779).Compounds 7-10 were prepared using slightly modified procedures. Allcompounds are >95% pure as determined by HPLC/MS and ¹H NMR.

Bacterial Strains.

UTI189 is a prototypical cystitis isolate of serotype O18:K1:H7. PBC-1is a TMP-SMZ^(R) strain of serotype OX13:K1:H10 isolated from a 59 yearold asymptomatic female with a history of recurrent UTI and diagnosis ofprimary biliary cirrhosis.

Synthesis of Mannosides.

1. General Synthesis, Purification, and Analytical Chemistry Procedures.

Starting materials, reagents, and solvents were purchased fromcommercial vendors unless otherwise noted. ¹H NMR spectra were measuredon a Varian 300 MHz NMR instrument. The chemical shifts were reported as6 ppm relative to TMS using residual solvent peak as the referenceunless otherwise noted. The following abbreviations were used to expressthe multiplicities: s=singlet; d=doublet; t=triplet; q=quartet;m=multiplet; br=broad. High-performed liquid chromatography (HPLC) wascarried out on GILSON GX-281 using Waters C18 5 μM, 4.6*50 mm and WatersPrep C18 5 μM, 19*150 mm reverse phase columns, eluted with a gradientsystem of 5:95 to 95:5 acetonitrile:water with a buffer consisting of0.05% TFA. Mass spectra (MS) were performed on HPLC/MSD usingelectrospray ionization (ESI) for detection. All reactions weremonitored by thin layer chromatography (TLC) carried out on Merck silicagel plates (0.25 mm thick, 60F254), visualized by using UV (254 nm) ordyes such as KMnO₄, p-Anisaldehyde and CAM. Silica gel chromatographywas carried out on a Teledyne ISCO CombiFlash purification system usingpre-packed silica gel columns (12 g˜330 g sizes). All compounds used forbiological assays are greater than 95% purity based on NMR and HPLC byabsorbance at 220 nm and 254 nm wavelengths.

2. Experimental Procedure for the Preparation of Mannoside 8 2.1[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate

Under nitrogen atmosphere and at room temperature, boron trifluoridediethyl etherate (3.41 g, 24 mmol) was added dropwise into the solutionof α-D-mannose pentaacetate (3.12 g, 8 mmol) and 4-bromo-2-methylphenol(2.99 g, 16 mmol) in 100 ml of anhydrous CH₂Cl₂. After a few mins themixture was heated to reflux and kept stirring for 45 hrs. The reactionwas then quenched with water and extracted with CH₂Cl₂. The CH₂Cl₂ layerwas collected dried with Na₂SO₄, concentrated. The resulting residue waspurified by silica gel chromatography with hexane/ethyl acetatecombinations as eluent, giving the title compound (3.22 g) in 77% yield.¹H NMR (300 MHz, CHLOROFORM-d) δ 7.18-7.38 (m, 2H), 6.97 (d, J=8.79 Hz,1H), 5.50-5.59 (m, 1H), 5.43-5.50 (m, 2H), 5.32-5.42 (m, 1H), 4.28 (dd,J=5.63, 12.50 Hz, 1H), 3.99-4.15 (m, 2H), 2.27 (s, 3H), 2.20 (s, 3H),2.02-2.11 (three singlets, 9H); MS (ESI): found: [M+Na]+, 539.0.

2.2N-methyl-3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide(8)

Under nitrogen atmosphere, the mixture of[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate(0.517 g, 1 mmol), 3-(N-Methylaminocarbonyl)phenylboronic acid pinacolester (0.392 g, 1.5 mmol), cesium carbonate (0.977 g, 3 mmol) andtetrakis(triphenylphosphine)palladium (0.116 g, 0.1 mmol) indioxane/water (15 mL/3 mL) was heated at 80° C. with stirring for 1 hunder a nitrogen atmosphere. After cooling to RT, the mixture wasfiltered through silica gel column to remove the metal catalyst andsalts with hexane/ethyl acetate combinations as eluent. The filtrate wasconcentrated, and then dried in vacuo. The residue was diluted with 15mL of methanol containing a catalytic amount of sodium methoxide (0.02M) and the mixture was stirred at RT overnight. H+ exchange resin (DOWEX50WX4-100) was added to neutralize the mixture. The resin was filteredoff and the filtrate was concentrated. The resulting residue waspurified by silica gel chromatography with CH₂Cl₂/MeOH combinations aseluent, giving the title compound (0.260 g) in 64% yield for two steps.¹H NMR (300 MHz, METHANOL-d4) δ 7.94 (t, J=1.65 Hz, 1H), 7.57-7.72 (m,2H), 7.33-7.50 (m, 3H), 7.23 (d, J=8.52 Hz, 1H), 5.48 (d, J=1.92 Hz,1H), 4.00 (dd, J=1.79, 3.43 Hz, 1H), 3.83-3.94 (m, 1H), 3.60-3.76 (m,3H), 3.46-3.58 (m, 1H), 2.87 (s, 3H), 2.24 (s, 3H). MS (ESI): found:[M+H]⁺, 404.2.

Mannosides 7 and 9 were prepared following a similar procedure to thesynthesis of 8.

3. Experimental Procedure for the Preparation of Mannoside 10 3.1[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-trifluoromethylphenoxy)tetrahydropyran-3-yl] acetate

Using the procedure outlined in 2.1 using4-bromo-2-trifluoromethylphenol, the title compound was obtained (2.5 g)in 54% yield. ¹H NMR (300 MHz, CHLOROFORM-d) b 7.75 (d, J=2.20 Hz, 1H),7.61 (dd, J=2.47, 8.79 Hz, 1H), 7.15 (d, J=8.79 Hz, 1H), 5.61 (d, J=1.65Hz, 1H), 5.32-5.58 (m, 3H), 4.28 (dd, J=5.22, 12.36 Hz, 1H), 3.95-4.22(m, 2H), 2.21 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H); MS(ESI): found: [M+Na]+, 593.0.

3.2N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-dicarboxamide

Dimethyl 5-bromobenzene-1,3-dicarboxylate (10.6 g, 36.8 mmol) wasdissolved in a 33 wt % solution of methylamine in EtOH (30 mL) andstirred for 6 h at RT. The precipitate that formed during the reactionwas filtered to give 5.3 g (53%) of the intermediate5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide as a white solid.Concentration of the remaining filtrate yielded an additional 4.6 g(46%) of product. 5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide (5.3g, 19.5 mmol), Pd(dppf)₂Cl₂ (0.87 g, 1.2 mmol), di-pinacolborane (6.1 g,24 mmol), and potassium acetate (7.8 g, 80 mmol) were dissolved in DMSO(100 mL). The solution was stirred under vacuum and then repressurizedwith nitrogen. This process was repeated 3 times and then the resultantmixture was stirred at 80° C. for 5 h under a nitrogen atmosphere. Afterremoval of the solvent under high vacuum, the crude material waspurified by silica gel chromatography to give the title compound as alight tan solid (2.2 g, 35%). ¹H NMR (300 MHz, DMSO-d6 δ 8.63 (m, 2H),8.41 (t, J=1.51 Hz, 1H), 8.23 (d, J=1.65 Hz, 2H), 2.80 (s, 3H), 2.78 (s,3H), 1.33 (s, 12H). MS (ESI): found: [M+H]⁺, 319.2.

3.3N1,N3-dimethyl-5-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamide(10)

Using the procedure outlined in 2.2 with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-trifluoromethyl-phenoxy)tetrahydropyran-3-yl]acetate (0.57 g) andN1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-dicarboxamide(0.48 g), the title compound was obtained (0.340 g) in 69% yield for thetwo steps. ¹H NMR (300 MHz, METHANOL-d4) δ 8.17-8.24 (m, 1H), 8.14 (d,J=1.65 Hz, 2H), 7.92 (d, J=1.92 Hz, 1H), 7.87 (dd, J=2.20, 8.79 Hz, 1H),7.57 (d, J=8.79 Hz, 1H), 5.64 (d, J=1.65 Hz, 1H), 4.04 (dd, J=1.65, 3.30Hz, 1H), 3.87-3.96 (dd, 1H), 3.64-3.83 (m, 3H), 3.48-3.63 (m, 1H), 2.93(s, 6H). MS (ESI): found: [M+H]+, 515.1.

Biofilm Assay.

UTI189 was grown in LB broth in wells of PVC microtiter plates at 23° C.in the presence of individual mannosides at varying concentrations.After 48 h of growth, wells were rinsed with water and stained withcrystal violet for quantification as described. For biofilm disruptionactivity in PVC plates, UTI189 was grown in LB broth in wells of PVCmicrotiter plates at 23° C. After 24 h of growth, mannoside was addedand biofilms were grown for an additional 16 h. Wells were then rinsed,stained with crystal violet and quantified. For biofilm disruptionactivity on PVC coverslips, UTI189 was grown in LB broth in 50 mLconicals containing PBC coverslips at 23° C. After 24 h of growth, 0.3μM ZFH-2056 was added and biofilm was grown for an additional 16 h.Coverslips were then rinsed, fixed with 2% paraformaldehyde (v/v),stained with SYTO9 (1:1000 in PBS; Molecular Probes) and observed with aZeiss LSM410 confocal laser scanning microscope under a 63× objective.

Animal Infections.

Bacteria were grown under type 1 pili-inducing conditions (2×24 h at 37°C. statically in LB). The bacteria were harvested and resuspended to anOD₆₀₀ of 0.5 in PBS. Eight-week-old C3H/HeN (Harlan) female mice wereanesthetized by inhalation of isoflurane and infected via transurethralcatheterization with 50 μl of the bacterial suspension, resulting in1-2×10⁷ inoculum. At 6 hpi, mice were sacrificed by cervical dislocationunder anesthesia and the bladders were immediately harvested andprocessed as described below. All animal studies using mice wereapproved by the Animal Studies Committee of Washington University(Animal Protocol Number 20100002).

Chronic Infection.

Mice were infected with UTI189 and the infection was allowed to continuefor 2 weeks. At 12 days post-infection, urine was collected and titeredto determine mice chronically infected (urine titers >10⁶). At 2 wpi,chronically infected mice were treated PO with 50 mg/kg or 100 mg/kg ofmannoside. 6 h posttreatment, mice were sacrificed and bladders wereaseptically removed and homogenized to determine tissue titers.

Enumeration of Bladder IBCs.

For animal pretreatment experiments, mannoside ZFH-2056 was administeredeither IP (5 mg/kg) or orally (100 mg/kg) 30 min prior to inoculationwith UTI89. To accurately count the number of IBCs, mice were sacrificed6 hpi and bladders were aseptically removed, bisected, splayed onsilicone plates and fixed in 2% paraformaldehyde (v/v). IBCs, readilydiscernable as punctate violet spots, were quantified by LacZ stainingof whole bladders.

Pharamacokinetic Analysis.

For intraperitoneal dosing, 50 μl of a 2 mg/ml (5 mg/kg) or 4 mg/ml (10mg/kg) solution of 6 in PBS was injected into the peritoneal cavity ofthe mouse. For oral dosing, 100 μl of a 20 mg/ml (100 mg/kg) solution ofZFH-2056 in 8% DMSO was inoculated with a gavage needle into the mousestomach. Urine was collected at 30 min, 1, 2, 3, 4, 6, and 8 hpost-treatment. An equal volume of 10 μM internal standard (ZFH-2050)was added to the urine. Mannosides were extracted from the urine byloading on C18 columns (100 mg, Waters), washing with 30% methanol, andeluting with 60% methanol. Vacuum-concentrated eluates were analyzedusing liquid chromatography-mass spectrometry system with a lower heatedcapillary temperature of 190° C. and a gradient as follows: Solvent B(80% acetonitrile in 0.1% formic acid) was held constant at 5% for 5minutes, increased to 44% B by 45 minutes, and then to a 95% B by 65minutes. SRM mode quantification was performed with collision gas energyof 30% for the following MS/MS transitions (precursor m/z/product m/z):compound 6, 447/285; compound 3, 390/228. Absolute quantification wasachieved by comparison to a calibration curve.

Bladder Tissue Bacterial Titer Determination.

Mannoside ZFH-2056 was administered either IP (5 mg/kg) or orally (100mg/kg) 30 min prior to inoculation with UTI89. To enumerate the bacteriapresent, mice were sacrificed at 6 hpi and bladders were asepticallyremoved and homogenized in 1 ml PBS, serially diluted and plated onto LBagar plates. CFU was enumerated after 16 h of growth at 37° C.

Confocal Microscopy.

Mannoside 6 was administered IP (5 mg/kg) 30 min prior to inoculationwith UTI89. To visualize bacterial behavior within the bladder, micewere sacrificed at 6 hpi and bladders were aseptically removed,bisected, splayed on silicone plates revealing the luminal surface andfixed in 2% paraformaldehyde (v/v). The splayed bladders were thenincubated for 20 min at room temperature with (i) SYTO9 (1:1000 in PBS;Molecular Probes) to stain bacteria and (ii) Alexa Fluor 594-conjugatedwheat germ agglutinin (WGA; 1:1000 in PBS; Molecular Probes) to stainthe bladder luminal surface. Bladders were rinsed with PBS, mountedusing Prolong Gold antifade reagent (Invitrogen) and examined with aZeiss LSM510 confocal laser scanning microscope under a 63× objective.SYTO9 and WGA were excited at 488 and 594 nm, respectively.

Gentamicin Protection Assay.

To enumerate bacteria present in the intracellular versus extracellularcompartments, bladders were aseptically harvested at 6 hpi. The bladderswere then bisected twice and washed three times in 500 μl of PBS each.The wash fractions were pooled, lightly spun at 500 rpm for 5 min topellet exfoliated bladder cells, serially diluted, and plated onto LBagar to obtain the luminal fraction. The bladders were treated with 100μg of gentamicin/ml for 90 min at 37° C. After treatment, the bladderswere washed twice with PBS to eliminate residual gentamicin, homogenizedin 1 ml of PBS, serially diluted, and plated onto LB agar to enumberatethe CFUs in the intracellular fraction.

Antibiotic Treatment.

Mice were given TMP-SMZ in the drinking water at a concentration of 54μg/ml and 270 μg/ml, respectively. Water was changed daily for 3 daysprior to inoculation with UTI89. Mice remained on TMP-SMZ during theinfection. To determine TMP-SMZ concentration in the urine, urine wascollected after 3 days of TMP-SMZ treatment and quantified by LC-MSfollowing addition of sulfisoxazole as an internal standard.

Growth Curve.

An overnight culture of PBC-1 was diluted 1:1000 in LB in the absence orpresence of TMP-SMZ and/or mannoside 6. The highest concentration ofTMP-SMZ used was 512 μg/ml and 2560 μg/ml, respectively. Two-folddilutions of TMP-SMZ were performed. Mannoside 6 was added at 100 μM.Growth curves were performed in a 96-well plate at 37° C. with A₆₀₀readings taken every 30 min for 8 h. Minimum inhibitory concentration(MIC) was calculated as the lowest concentration of antibiotic thatprevented growth of the bacterial strain.

Statistical Analysis.

Observed differences in bacterial titers and IBC numbers were analyzedfor significance using the nonparametric Mann-Whitney U test (Prizm;GraphPad Software)

Introduction for Examples 30-36.

FimH is a mannose-specific bacterial lectin located at the tip of type 1pili, an adhesive fiber produced by uropathogenic E. coli (UPEC). FimHis known to bind to mannosylated human uroplakins that coat the luminalsurface of the bladder and has also been shown to be involved ininvasion of human bladder cells and mast cells, triggering apoptosis andexfoliation and inducing elevated levels of cAMP. Furthermore, FimHrecognizes N-linked oligosaccharides on beta1 and alpha3 integrins,which are expressed throughout the urothelium. Murine uroplakin ishighly homologous to human and FimH has been shown to facilitatebacterial colonization and invasion of the bladder epithelium in murinemodels. Internalized UPEC are exocytosed in a TLR-4 dependent process;however, bacteria can escape into the host cell cytoplasm, where theyare able to subvert expulsion and innate defenses by aggregating intobiofilm-like intracellular bacterial communities (IBCs) in a FimHdependent process. Subsequently, UPEC disperse from the IBC, escape intothe bladder lumen, and re-initiate the process by binding and invadingnaive epithelial cells where they are able to establish quiescentintracellular reservoirs that can persist in a dormant state, tolerantto antibiotic therapy and subsequently serve as seeds for recurrentinfection. In humans, the severity of UTI was increased and theimmunological response was greater in children with infections caused bytype 1 piliated UPEC strains and type 1 pilus expression has been shownto be essential for UTI in mouse models. In addition, a recent studyconcluded that type 1 pili play an important role in human cystitis andit has been reported that type 1 pili fulfill “Molecular Koch'spostulates of microbial pathogenesis. In agreement with these findingsand in support of a role for FimH in humans, it has been shown that thefimH gene is under positive selection in human clinical isolates ofUPEC. Aspects of the UPEC pathogenic cascade extensively characterizedin a murine model of infection have been documented in samples fromhuman clinical studies such as filamentation and IBC formation. Targetedinhibitors of FimH adhesion which block both E. coli invasion andbiofilm formation thus hold promising therapeutic potential as newantibacterials for the treatment of UTI and the prevention ofrecurrence. The discovery of simple D-mannose derivatives as inhibitorsof bacterial adherence was first reported almost three decades ago butearly mannosides showed only weak inhibition of bacterial adhesion.Consequently, the vast majority of research in this area has beenfocused on multivalent mannosides, which have been pursued in an effortto improve binding avidity to type 1 pili, which can be present in largenumbers on a single bacterium (up to hundreds). While substantialprogress has been made with this approach, these high molecular weightstructures are not suitable for in vivo evaluation or clinicaldevelopment as oral drugs. The recent X-ray crystal structures ofD-mannose, butyl mannoside, and mannotriose bound to FimH have enabledthe rational structure-based design of tighter binding alkyl-, phenyl-and biphenyl-mannoside FimH inhibitors. The urgency for developing neworally bioavailable FimH inhibitors as a targeted strategy for thetreatment of UTI alternative to broad spectrum antibiotics is reinforcedby the rate of recurrence seen in these type of infections as well asincreasing clinical resistance of UPEC to first line antibiotictreatments.

Example 30. Tight Binding Ortho-Substituted Biphenyl Mannosides

A matched pair analysis was performed of monoester 1 compared toortho-substituted analogs bearing halogen and small alkyl groups shownin FIG. 47. The compounds were evaluated for their potency in thehemagglutination inhibition (HAI) assay and it was discovered that allbiphenyl ring substitutions yielded more potent inhibitors (Table 17).Ortho-CI mannoside 4b had a HAI Titer EC₉₀ of 30 nM which is more than30-fold better than matched pair 1 while the Me analog 4c was 8-foldmore active (HAI Titer EC₉₀=120 nM). Substitution with CF3 gave the mostpotent analog 4d with an HAI Titer EC₉₀ of 30 nM whereas the OMe (4e)and F (4a) analogs showed smaller improvements in activity following thetrend CF3>Cl=Me>OMe>F. This data suggests that increased hydrophobiccontact with the tyrosine gate and Ile52, or with Ile13 at the oppositeridge of the mannose binding pocket could explain this enhanced potencysince better activity correlates well with increased hydrophobicity asevidenced by the fact that fluoro analog 4a shows no improvement inactivity relative to unsubstituted matched pair 1 and thetrifluoromethyl analog 4c which has the largest hydrophobic surface areashows the highest activity. However, it is possible that the orientationof both phenyl rings are altered slightly and are restricted to aconformation more conducive to improved FimH binding with Tyr137, Tyr48,Ile52, Ile13 and/or Arg98 residues.

TABLE 17 Potency enhancement and PAMPA data from ortho-substitution ofbiphenyl mannosides. HAI Biofilm Titer Prevention PAMPA EC_(>90) IC₅₀ MWLogP_(e) Compound (μM) (μM) (g/mol) PSA CLogD_(7.4) (cm²/sec) 1 (ester)1.00 0.94 390.4 126 1.17 −5.42 4a (F) 0.75 408.4 126 1.00 4b (Cl) 0.030.26 424.8 126 1.64 −4.29 4c (Me) 0.12 0.33 404.4 126 1.82 4d (CF₃) 0.030.17 458.4 126 1.62 −3.91 4e (OMe) 0.19 0.89 420.4 135 0.47 6 (m-CO₂Me)1.0 448.4 152 0.93 −4.08 2 (amide) 0.50 1.35 389.4 128 0.28 5a (Cl) 0.120.52 423.8 128 0.75 5b (Me) 0.06 0.16 403.4 128 0.93 −3.89 5c (CF₃) 0.030.13 457.4 128 0.73 3 (di-amide) 0.37 0.74 446.4 158 0.71 −4.51 7 (CF₃)0.01 0.043 514.5 158 1.15 −6.27 8 (Me) 0.02 0.073 460.5 158 1.35 −8.46 1(ester) 1.00 0.94 390.4 126 1.17 −5.42 4a (F) 0.75 408.4 126 1.00 4b(Cl) 0.03 0.26 424.8 126 1.64 −4.29 4c (Me) 0.12 0.33 404.4 126 1.82 4d(CF₃) 0.03 0.17 458.4 126 1.62 −3.91 4e (OMe) 0.19 0.89 420.4 135 0.47 6(m-CO₂Me) 1.0 448.4 152 0.93 −4.08 2 (amide) 0.50 1.35 389.4 128 0.28 5a(Cl) 0.12 0.52 423.8 128 0.75 5b (Me) 0.06 0.16 403.4 128 0.93 −3.89 5c(CF₃) 0.03 0.13 457.4 128 0.73 3 (di-amide) 0.37 0.74 446.4 158 0.71−4.51 7 (CF₃) 0.01 0.043 514.5 158 1.15 −6.27 8 (Me) 0.02 0.073 460.5158 1.35 −8.46

The outcome of this preliminary study directed the investigation ofanalogs which were more metabolically stable and soluble than esterssuch as amides 5a-c (FIG. 47 and Table 17). A similar trend was observed(CF3>Me>Cl) with CF3 amide 5c having the best activity but in this casethe Me analog 5b showed better activity than Cl analog 5a. Analogs werealso explored with substitution on the meta position, exemplified byester 6, which retains potency relative to 1 but did not lead to anyenhancement. Ortho-CF3 7 and Me 8 diamide matched pairs to original leadcompound 3 were next developed which were exponentially more potent thanany previously reported mannoside FimH inhibitors with an HAI Titer EC₉₀of 8 nM and 16 nM, respectively. This unprecedented level of cellularactivity corresponds to a 200,000-fold improvement over α-D-mannose anda 15,000-fold improvement over an early reported inhibitorbutyl-α-D-mannoside (HAI Titer EC90=125 μM) and 50-fold better thanprevious lead compound 3.

The biofilm inhibition assay was utilized to test these mannosides'ability to prevent bacteria from forming IBCs, a critical pathogenicprocess in the development of UTIs. As shown in Table 17, the BiofilmPrevention IC₅₀s correlate quite well with the potencies determined bythe HAI assay. Introduction of an ortho-substituent (e.g. methyl) to thebiphenyl mannoside improved the biofilm activity by 8-fold frommannoside 2 (IC₅₀=1.35 μM) to mannoside 5b (IC₅₀=0.16 μM). This dataconfirmed the mannoside's functional effect and activity on UPEC derivedfrom FimH inhibition with a secondary assay. Biofilm IC50s were utilizedin conjunction with pharmacokinetic parameters as a key measure of thepredicted lowest effective mannoside concentration in the urine requiredfor efficacy in vivo to be discussed vide infra.

Mannosides were synthesized by traditional Lewis acid mediatedglycosylation of mannose penta-acetate by reaction with 2-substituted4-bromophenols using BF3 etherate (Scheme 1). Suzuki cross-coupling withcommercially available 3-substituted phenyl boronic acid derivativesgave protected ortho-substituted 4′-biphenyl mannosides in excellentyields and subsequent deprotection with NaOMe gave mannosides 4-5. 6 wasprepared following the procedure previously described. Synthesis ofdi-amides 7-8 followed a similar procedure but required the synthesis of3,5-di-(N-methyl aminocarbonyl)phenyl boronic acid pinacol ester 9.Shown in Scheme 1, amidation of dimethyl 5-bromoisophthalate by reactionof methylamine in ethanol proceeded in quantitative yield to giveN,N-dimethyl 5-bromoisophthalamide. Installation of the boronate esterwas accomplished by Pd-mediated coupling with bis(pinacolato)diboron togive 9. Suzuki coupling and deprotection as before yielded compounds7-8.

Example 31. Mannosides with Increased pKa Functionality

To help improve tissue penetration and exposure of mannosides in thebladder, amide derivatives were explored containing functional groupswith higher pKa (Table 18). Higher pKa compounds containing basicmoieties such as amines tend to have increased tissue penetration.Therefore, mannosides 10a-f were prepared via standard HATU-mediatedcoupling reaction of 4′-(α-D-mannopyranosyloxy)biphenyl-3-carboxylicacid with various amines. Unexpectedly, aminoethyl amide 10b showed adisappointing 6-fold drop in activity relative to methyl amide 2.However, hydroxyethyl amide 10a was equipotent to 2. It was also foundthat more hydrophobic tertiary amide piperazine analogs 10c and 10d hadlargely decreased potency (HAI EC₉₀=4 μM). Interestingly, pyridyl amides10e and 10f showed slightly improved activity most pronounced with4-pyridyl derivative 10e. While it is unclear to the reason fordecreased potency of higher pKa substituents it is plausible that thiseffect could originate from charge-charge repulsion with Arg98 sidechain at the edge of FimH binding pocket.

TABLE 18 Exploration of amide substitution with increased pKa

HAI Titer Compound R EC_(>90) (μM) 10a

0.50 10b

3.0 10c

4.0 10d

4.0 10e

0.25 10f

0.37

Example 32. Mannosides with Heterocyclic Replacements

Heterocyclic replacements of the terminal biphenyl ring were thenpersued as an alternate approach to improve the drug-like properties oflead mannosides. In target compounds the key H-bond donor to Arg98 ofFimH was retained so we could directly compare the effects ofheterocyclic ring replacement. In order to synthesize a library ofheterocycles in a divergent fashion, a new Suzuki synthesis wasdeveloped using a 4-mannopyranosyloxyphenyl boronate intermediate 11 inplace of 4-bromophenyl-α-D-mannoside. Only a limited number ofheteroaryl boronates are commercially available and this new methodologyallows for the use of more readily obtainable heteroaryl bromides ascoupling partners. All heteroaryl bromides used in Table 19 werecommercially available with reasonable prices except bromothiophenederivatives 16 and 17 (Scheme 2). 16 was prepared via Curtius reactionby first treatment of 5-bromothiophene-3-carboxylic acid withdiphenylphosphoryl azide (DPPA) to form isocyanate intermediate, thenquenching with ammonia. 17 was synthesized according to previous method.As shown in Scheme 2, Starting from 4-bromophenyl-α-D-mannoside,bis(pinacolato)diboron and Pd(dppf)Cl₂ in DMSO, intermediate 11 wasprepared in good yield. Suzuki cross coupling of 11 with various arylbromides, followed by acetyl deprotection gave target compounds.Compounds synthesized, shown in Table 19, include pyridyl esters 12a and12b which showed excellent activity in the HAI titer with improvementover phenyl ester 1. Furthermore, ring replacement with a thiophene ureacarboxylate led to dramatic advancements in FimH activity as exemplifiedby compound 13a which has an HAI EC₉₀=16 nM. In order to ascertainwhether the carboxylate ester group or urea were responsible for thislarge increase in potency, ester 13b and urea 13c were synthesized todiscover that the enhancement results from a combined effect of bothfunctional since 13b or 13c have much decreased activity. It is unknownwhy there is a synergistic effect from the compound with both urea andcarboxylate but from previous work on thiophene carboxamide ureas it wasshown that an intramolecular H-bond exists between the internal NH ofthe urea and the carbonyl of the ester. This conformational restrictionmight enhance binding entropically to FimH likely from the ureacarbonyl. Several fused rings were also persued such as isoquinolinederivatives 14 and 15 to examine the effects of isosteric replacementfor the aryl carbonyl H-bond acceptor where the heterocyclic ringnitrogen is designed to accept a H-bond from the FimH Arg98 side chain.The promising HAI assay results for 14a-b to 15a-b clearly provides muchevidence that the orientation of the C═N in 15a or C═O in 15b is likelythe same as the conformation of C═O of mannoside 1 in crystal structureof FimH-mannoside 1²⁵ bringing the potency up by as much as 10-fold overmannoside 1.

TABLE 19 Heterocyclic modifications to biphenyl ring for improvingdrug-like properties HAI Titer Compound R EC_(>90) (μM) 12a

 0.50 12b

 0.19 13a

 0.02 13b

1.0 13c

1.0 13d

2.0 14a

 0.75 14b

 0.38 15a

 0.25 15b

 0.10

Example 33. Direct Binding of FimH-Mannoside

In order to better understand how the excellent potency in cell-basedHAI and biofilm assays is correlated with FimH binding by biarylmannosides and to more precisely select the best lead compounds for invivo pharmacokinetic (PK) studies, a biolayer interferometry method wasdeveloped to directly measure the binding affinities (K_(d)) of FimHinhibitors. As shown in Table 20, earlier mannosides 19 33 showingmoderate potency in the HAI assay had K_(d) values as low as 110 nM.Strikingly, for compounds 1 and 2, K_(d) values were in the picomolarrange. For mannosides 5, 7, and 8, however, K_(d) could not becalculated because the off-rates were too low to measure. In order toovercome this obstacle, differential scanning fluorimetry (DSF) wasutilized to rank the high-affinity mannosides. DSF measures the meltingtemperature change of protein when binding to small molecules. Meltingtemperature shifts are proportional to the free energy of binding, andmelting temperatures increase even as ligand concentration exceeds theK_(d). As illustrated in Table 20 and FIG. 48, the melting temperatureof FimH without mannoside was about 60° C. and rose to between 68° C.and 74° C. when binding to mannosides 19˜33 of moderate potency. Withtight binding mannosides 5b, 7, 8 the melting temperature of FimH rangedfrom 74° C. to 76° C., suggesting that improved mannosides likely bindFimH with low picomolar affinity. FIG. 48 illustrates that DSF ranksmannosides in a similar fashion to the HAI assay except for 5c and 25,demonstrating that this is a general and reliable method toqualitatively rank FimH-mannoside binding when K_(d)s span many ordersof magnitude. Thus, these direct FimH binding studies solidified thatthe high potencies stemming from mannosides 5b, 7, 8 derives directlyfrom extremely tight binding to the FimH lectin and not othernon-specific effects from the cell assays.

TABLE 20 Results of Octet assay and DSF assay                      Compound

                    HAI Titer EC_(>90) (μM)                   OCtetK_(d) (nM)                 DSF Melting Temp. (° C.)  7

 0.008 N.D.* 76.15  8

 0.016 N.D.* 75.76  5c

 0.032 N.D.* 72.29  5b

 0.060 N.D.* 74.46 18

 0.150 N.D.* 72.53 10f

 0.375 N.D.* 72.53  3

 0.375  0.14 74.39 12a

 0.500 N.D.* 73.68  2

 0.500  0.01 73.38 19

 1.000  0.49 72.05  1

 1.000  0.08 73.39 20

 1.500  1.25 71.77 21

 2.000 N.D.* 70.72 22

 2.000 14.5  72.39 23

 2.000  3.45 73.25 24

 2.000  2.00 70.90 25

 2.000  1.56 68.32 26

 2.000  1.14 72.30 27

 2.000  1.00 71.69 28

 3.000  2.61 71.16 29

 4.000  3.99 68.69 30

 4.000  2.21 70.93 31

 6.000  2.72 70.75 32

 6.000  2.72 70.75 33

 8.000  3.00 71.17 Phenyl-α-D- mannoside

30.000 N.D.* 68.93 34

60.000 43.66 68.11 *└N.D. = not determined

An electrostatic surface was generated with the most potent mannoside 7docked to FimH shown in FIG. 49. The large boost in binding affinity toFimH can be attributed to the fact that in the model the orthotrifluoromethyl group orients directly at Ile13 resulting in a verystrong hydrophobic interaction with FimH. The added hydrophobicityencompassed by the fluorine atoms in mannoside 7 results in furtheraugmented binding to FimH relative to ortho methyl mannoside 8.

Example 34. Stability and Elimination Kinetics of Mannoside

Mannoside 3 shows efficacy in vivo in the treatment and prevention ofestablished UTIs in mice when dosed orally but the compound displayedsome metabolic instability from hydrolysis of the glycosidic bond (FIG.50A) and very rapid elimination kinetics to the urine partly due to itslow C Log D value. While renal clearance is an attractive feature forUTI therapy, improved mannosides were desirable encompassing lowerclearance rates as well as increased oral bioavailability and bladdertissue permeability. In order to have a general idea of the eliminationrate of mannoside 3, pharmacokinetic (PK) studies of its urinaryclearance were conducted in mice (FIG. 50B). These experimentsdemonstrated that the lower oral dosing of 20 mg/kg was unlikely tomaintain effective concentrations (as determined by Biofilm IC₅₀) due torapid clearance. Maintenance of mannoside 3 levels above the minimaleffective level of 0.74 μM during in an eight-hour period required alarger, 100 mg/kg dose. During the PK study, a small amount of aphenolic biphenyl metabolite (R) (FIG. 50A) was detected in the urineindicating some glycoside bond hydrolysis takes place upon oral dosing.Urine levels of the R group were unchanged from the 100 and 200 mg/kgdoses, suggesting that metabolism by glycoside bond hydrolysis issaturated between the 20 and 100 mg/kg doses (FIG. 50B).

Example 35. Parallel Artificial Membrane Permeability Assay (PAMPA)

The low Log D of polyhydroxylated sugar-based mannosides and othercarbohydrate-derived compounds can limit their ability to cross cellmembranes in the absence of active transport mechanisms and soincreasing their hydrophobicity is one strategy to help improve cellpermeability and oral bioavailability of this class of molecules. Theortho-substituted mannosides described above were designed for increasedhydrophobic contact with FimH but also in part to increase the Log D andwere predicted to improve the solubility, oral bioavailability andbladder tissue penetration relative to starting mannosides 1-3. It wasanticipated that the newly designed inhibitors would also have increasedmetabolic stability via protection of the glycosidic bond from acidichydrolysis in the gut and enzymatic hydrolysis by α-mannosidases inblood and tissues. In order to test these hypotheses experimentally, theParallel Artificial Membrane Permeability Assay (PAMPA) was used. PAMPAis commonly used as an in vitro model of passive, transcellularpermeability to predict oral bioavailability for drug candidates. Themost potent biphenyl mannosides was tested in this model forprioritizing compounds to evaluate further in animal PK and efficacystudies. Shown in Table 20, Compound 5b with Log P_(e) of −3.89 cm²/secproved to have the highest oral absorption, while the most potentmannosides (determined by HAI assay) 7 and 8 with Log P_(e) of −6.27 and−8.46 cm²/sec exhibited significantly lower oral bioavailability.

Example 36. Pharmacokinetic Studies for Selected Mannoside Inhibitors

Based on these results, oral PK studies were performed in mice to assessany improvements in the PK of these ortho-substituted mannosides.Compounds were dosed at 50 mg/kg and plasma and urine samples were takenat 30 min and 1, 2, 3, 4, 6 hours after dosing. As demonstrated in FIG.51, a generally 10-fold higher mannoside concentration was observed inurine (FIG. 51B) compared to plasma (FIG. 51A), indicating a highclearance rate for these mannosides, which in this case aids in clearinguropathogens on the bladder surface. It was found that compounds 8 and5b consistently maintain a high level of concentration in both urine andplasma, which is well above the predicted minimum effectiveconcentration within a 6-hour period. While 100 mg/kg dosing ofmannoside 3 was required to achieve effective mannoside concentrations,only 50 mg/kg dosing of mannoside 5b was required, permitting a muchlarger therapeutic window for treatment. Taken together with PAMPAresults, high oral bioavailability and in vivo efficacy in recentlyreported animal studies support mannoside 5b as the most promisingtherapeutic candidate for UTI treatment/prevention.

Conclusions for Examples 30-36

Using a combination of traditional ligand-based and X-raystructure-guided approaches with structure-activity relationship (SAR)driven by cell-based hemagglutination and biofilm assays in combinationwith direct FimH binding assays, a diverse array of biaryl mannosideFimH inhibitors that exhibit affinities into the picomolar range wereidentified. While it was found the most potent mannoside 7 with respectto FimH binding affinity and activity in cell assays contains anortho-trifluoromethyl group off the phenyl ring adjacent to the mannosering group, the most promising inhibitor from in vivo studies is theortho-methyl analog 8 showing prolonged compound exposure in plasma andurine PK studies. A variety of heterocyclic biaryl mannosides thateither retain or improve FimH binding activity were also discovered. Thenovel inhibitors of UPEC type 1 mediated bacterial adhesion reportedherein show unprecedented activity in hemagglutination and biofilm invitro assays in addition to desirable pharmacokinetic properties invivo. Further optimization of lead mannosides is currently being focusedon the identification of mannose modifications with reduced sugar-likecharacter. Biaryl mannosides have high potential as innovativetherapeutics for the clinical treatment and prevention of urinary tractinfections. The unique mechanism of action of targeting the pilus tipadhesin, FimH, circumvents the conventional requirement for drugpenetration of the outer membrane and the potential for development ofresistance by porin mutations, efflux or degradative enzymes, allmechanisms that promote resistance to antibiotics. Current efforts aredirected at the selection of one or more clinical candidate drugsthrough rigorous preclinical evaluation in several models of recurrentUTI with antibiotic resistant forms of UPEC. These preclinical modelswill facilitate further optimization of current lead compounds.

Experimental Section for Examples 30-36

General Synthesis, Purification, and Analytical Chemistry Procedures.

Starting materials, reagents, and solvents were purchased fromcommercial vendors unless otherwise noted. ¹H NMR spectra were measuredon a Varian 300 MHz NMR instrument. The chemical shifts were reported asb ppm relative to TMS using residual solvent peak as the referenceunless otherwise noted. The following abbreviations were used to expressthe multiplicities: s=singlet; d=doublet; t=triplet; q=quartet;m=multiplet; br=broad. High-performed liquid chromatography (HPLC) wascarried out on GILSON GX-281 using Waters C18 5 μM, 4.6*50 mm and WatersPrep C18 5 μM, 19*150 mm reverse phase columns, eluted with a gradientsystem of 5:95 to 95:5 acetonitrile:water with a buffer consisting of0.05% TFA. Mass spectra (MS) were performed on HPLC/MSD usingelectrospray ionization (ESI) for detection. All reactions weremonitored by thin layer chromatography (TLC) carried out on Merck silicagel plates (0.25 mm thick, 60F254), visualized by using UV (254 nm) ordyes such as KMnO₄, p-Anisaldehyde and CAM. Silica gel chromatographywas carried out on a Teledyne ISCO CombiFlash purification system usingpre-packed silica gel columns (12 g˜330 g sizes). All compounds used forbiological assays are greater than 95% purity based on NMR and HPLC byabsorbance at 220 nm and 254 nm wavelengths.

Procedures for the Preparation of Biphenyl Mannoside Derivatives ThroughSuzuki Coupling Reaction with Bromophenyl Mannoside Derivatives asIntermediateN-methyl-3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide(5b).[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate

Under nitrogen atmosphere and at room temperature, boron trifluoridediethyl etherate (3.41 g, 24 mmol) was added dropwise into the solutionof α-D-mannose pentaacetate (3.12 g, 8 mmol) and 4-bromo-2-methylphenol(2.99 g, 16 mmol) in 100 ml of anhydrous CH₂Cl₂. After a few mins themixture was heated to reflux and kept stirring for 45 hrs. The reactionwas then quenched with water and extracted with CH₂Cl₂. The CH₂Cl₂ layerwas collected dried with Na₂SO₄, concentrated. The resulting residue waspurified by silica gel chromatography with hexane/ethyl acetatecombinations as eluent, giving[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate (3.22 g) in 77% yield. ¹H NMR (300 MHz, CHLOROFORM-d) δ ppm7.18-7.38 (m, 2H), 6.97 (d, J=8.79 Hz, 1H), 5.50-5.59 (m, 1H), 5.43-5.50(m, 2H), 5.32-5.42 (m, 1H), 4.28 (dd, J=5.63, 12.50 Hz, 1H), 3.99-4.15(m, 2H), 2.27 (s, 3H), 2.20 (s, 3H), 2.02-2.11 (three singlets, 9H). MS(ESI): found: [M+Na]+, 539.0.

N-methyl-3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide(5b)

Under nitrogen atmosphere, the mixture of[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate (0.517 g, 1 mmol), 3-(N-methylaminocarbonyl)phenylboronic acidpinacol ester (0.392 g, 1.5 mmol), cesium carbonate (0.977 g, 3 mmol)and tetrakis(triphenylphosphine)palladium (0.116 g, 0.1 mmol) indioxane/water (15 mL/3 mL) was heated at 80° C. with stirring for 1 hunder a nitrogen atmosphere. After cooling to RT, the mixture wasfiltered through silica gel column to remove the metal catalyst andsalts with hexane/ethyl acetate combinations as eluent. The filtrate wasconcentrated, and then dried in vacuo. The residue was diluted with 15mL of methanol containing a catalytic amount of sodium methoxide (0.02M) and the mixture was stirred at RT overnight. ^(H) exchange resin(DOWEX 50WX4-100) was added to neutralize the mixture. The resin wasfiltered off and the filtrate was concentrated. The resulting residuewas purified by silica gel chromatography with CH₂Cl₂/MeOH combinationsas eluent, giving the title compound (0.260 g) in 64% yield for twosteps. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm 7.94 (t, J=1.65 Hz, 1H),7.57-7.72 (m, 2H), 7.33-7.50 (m, 3H), 7.23 (d, J=8.52 Hz, 1H), 5.48 (d,J=1.92 Hz, 1H), 4.00 (dd, J=1.79, 3.43 Hz, 1H), 3.83-3.94 (m, 1H),3.60-3.76 (m, 3H), 3.46-3.58 (m, 1H), 2.87 (s, 3H), 2.24 (s, 3H). MS(ESI): found: [M+H]⁺, 404.2.

Methyl3-[3-fluoro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4a).[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-fluoro-phenoxy)tetrahydropyran-3-yl]acetate

it was prepared using the same procedure as for[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate in the synthesis of 5b. Yield: 25%. ¹H NMR (300 MHz,CHLOROFORM-d) δ ppm 7.30 (dd, J=2.34, 10.03 Hz, 1H), 7.21 (td, J=1.79,8.79 Hz, 1H), 7.08 (t, J=8.52 Hz, 1H), 5.48-5.58 (m, 2H), 5.46 (d,J=1.65 Hz, 1H), 5.31-5.41 (m, 1H), 4.23-4.31 (m, 1H), 4.13-4.22 (m, 1H),4.05-4.13 (m, 1H), 2.20 (s, 3H), 2.02-2.08 (three singlets, 9H). MS(ESI): found: [M+Na]+, 543.0.

Methyl3-[3-fluoro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4a)

4a was prepared using the same procedure as for 5b with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-fluoro-phenoxy)tetrahydropyran-3-yl]acetate and 3-methoxycarbonylphenyl boronic acid as the reactants.Yield: 66%. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm 8.21 (t, J=1.65 Hz, 1H),7.99 (td, J=1.44, 7.83 Hz, 1H), 7.84 (ddd, J=1.10, 1.92, 7.69 Hz, 1H),7.35-7.62 (m, 4H), 5.55 (d, J=1.92 Hz, 1H), 4.10 (dd, J=1.79, 3.43 Hz,1H), 3.94 (s, 3H), 3.86-4.00 (m, 1H), 3.59-3.86 (m, 4H). MS (ESI): found[M+Na]⁺, 431.1.

Methyl3-[3-chloro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4b).[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-chloro-phenoxy)tetrahydropyran-3-yl]acetate

it was prepared using the same procedure as for[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate in the synthesis of 5b. Yield: 46%. ¹H NMR (300 MHz,CHLOROFORM-d) δ ppm 7.55 (d, J=2.47 Hz, 1H), 7.33 (dd, J=2.47, 8.79 Hz,1H), 7.06 (d, J=8.79 Hz, 1H), 5.58 (dd, J=3.02, 10.16 Hz, 1H), 5.52 (s,1H), 5.49-5.54 (m, 1H), 5.33-5.42 (m, 1H), 4.22-4.32 (m, 1H), 4.04-4.17(m, 2H), 2.21 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H). MS(ESI): found [M+Na]⁺, 561.0.

Methyl3-[3-chloro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4b). 4b was prepared using the same procedure as for 5b with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-chloro-phenoxy)tetrahydropyran-3-yl]acetate and 3-methoxycarbonylphenyl boronic acid as the reactants. Itwas further purified by HPLC (C18, 15*150 mm column; eluent:acetonitrile/water (0.1% TFA)). Yield: 43%. ¹H NMR (300 MHz,METHANOL-d₄) δ ppm 3.59-3.71 (m, 1H) 3.71-3.85 (m, 3H) 3.94 (s, 3H) 4.01(dd, J=9.34, 3.30 Hz, 1H) 4.12 (dd, J=3.30, 1.92 Hz, 1H) 5.61 (d, J=1.65Hz, 1H) 7.40-7.49 (m, 1H) 7.49-7.62 (m, 2H) 7.68 (d, J=2.20 Hz, 1H) 7.82(ddd, J=7.76, 1.85, 1.10 Hz, 1H) 7.98 (dt, J=7.83, 1.30 Hz, 1H)8.14-8.25 (m, 1H). MS (ESI): found [M+H]⁺, 425.1.

Methyl3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4c)

4c was prepared using the same procedure as for 5b with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetate and 3-methoxycarbonylphenyl boronic acid as the reactants.Yield: 54%. H NMR (300 MHz, METHANOL-d₄) δ ppm 8.20 (t, J=1.51 Hz, 1H),7.94 (td, J=1.41, 7.90 Hz, 1H), 7.77-7.87 (m, 1H), 7.52 (t, J=7.55 Hz,1H), 7.39-7.48 (m, 2H), 7.27-7.38 (m, 1H), 5.56 (d, J=1.65 Hz, 1H), 4.08(dd, J=1.92, 3.30 Hz, 1H), 3.94-4.01 (m, 1H), 3.90-3.94 (m, 3H),3.68-3.83 (m, 3H), 3.55-3.65 (m, 1H), 2.31 (s, 3H). MS (ESI): found[M+Na]⁺, 427.1.

Methyl3-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4d).[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-bromo-2-(trifluoromethyl)phenoxy]tetrahydropyran-3-yl]acetate: it was prepared using the same procedure as for[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl]acetatein the synthesis of 5b. Yield: 54%. ¹H NMR (300 MHz, CHLOROFORM-d) b ppm7.74 (d, J=2.20 Hz, 1H), 7.61 (dd, J=2.33, 8.93 Hz, 1H), 7.15 (d, J=8.79Hz, 1H), 5.61 (d, J=1.92 Hz, 1H), 5.48-5.56 (m, 1H), 5.33-5.48 (m, 2H),4.21-4.34 (m, 1H), 3.97-4.14 (m, 2H), 2.21 (s, 3H), 1.99-2.12 (threesinglets, 9H). MS (ESI): found [M+Na]⁺, 593.0.

Methyl3-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4d). 4d was prepared using the same procedure as for 5b with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-bromo-2-(trifluoromethyl)phenoxy]tetrahydropyran-3-yl]acetate and 3-methoxycarbonylphenyl boronic acid as the reactants.Yield: 53%. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm 8.23 (t, J=1.51 Hz, 1H),8.01 (td, J=1.37, 7.69 Hz, 1H), 7.80-7.93 (m, 3H), 7.52-7.67 (m, 2H),5.66 (d, J=1.65 Hz, 1H), 4.07 (dd, J=1.79, 3.43 Hz, 1H), 3.95 (s, 3H),3.90-4.00 (m, 1H), 3.66-3.87 (m, 3H), 3.52-3.66 (m, 1H). MS (ESI): found[M+Na]+, 481.0.

Methyl3-[3-methoxy-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate(4e)

4e was prepared using the same procedure as for 5b. In the first step ofglycosidation reaction 4-bromo-2-methoxyphenol was used as glycosidationacceptor and in the second step of Suzuki coupling reaction3-methoxycarbonylphenyl boronic acid was used instead. All intermediateswere directly taken to the next step reaction without furtherpurification. 4e was further purified by HPLC (C18, 15*150 mm column;eluent: acetonitrile/water (0.1% TFA)). Yield: 3%. ¹H NMR (300 MHz,METHANOL-d₄) δ ppm 8.21 (t, J=1.51 Hz, 1H), 7.96 (td, J=1.44, 7.83 Hz,1H), 7.84 (ddd, J=1.24, 1.92, 7.83 Hz, 1H), 7.49-7.61 (m, 1H), 7.30 (d,J=8.24 Hz, 1H), 7.24 (d, J=1.92 Hz, 1H), 7.13-7.21 (m, 1H), 5.47 (d,J=1.92 Hz, 1H), 4.11 (dd, J=1.79, 3.43 Hz, 1H), 3.94 (s, 3H), 3.92 (s,3H), 3.86-4.03 (m, 1H), 3.67-3.86 (m, 4H). MS (ESI): found [M+Na]⁺,443.1.

3-[3-Chloro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]-N-methyl-benzamide(5a). 5a was prepared using the same procedure as for 5b. Yield: 59%. ¹HNMR (300 MHz, METHANOL-d₄) δ ppm 8.03 (t, J=1.51 Hz, 1H), 7.69-7.83 (m,3H), 7.32-7.67 (m, 3H), 5.60 (d, J=1.65 Hz, 1H), 4.12 (dd, J=1.79, 3.43Hz, 1H), 4.01 (dd, J=3.57, 9.34 Hz, 1H), 3.69-3.84 (m, 3H), 3.61-3.69(m, 1H), 2.95 (s, 3H). MS (ESI): found [M+Na]⁺, 424.1.

N-Methyl-3-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide(5c)

5c was prepared using the same procedure as for 5b. Yield: 65%. ¹H NMR(300 MHz, METHANOL-d₄) b ppm 7.98 (t, J=1.65 Hz, 1H), 7.77-7.87 (m, 2H),7.65-7.77 (m, 2H), 7.42-7.60 (m, 2H), 5.58 (d, J=1.65 Hz, 1H), 3.99 (dd,J=1.79, 3.43 Hz, 1H), 3.81-3.91 (m, 1H), 3.59-3.80 (m, 3H), 3.45-3.58(m, 1H), 2.87 (s, 3H). MS (ESI): found [M+H]⁺, 458.1.

Methyl2-(3-methoxycarbonylphenyl)-5-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-benzoate(6). Methyl 5-hydroxy-2-(3-methoxycarbonylphenyl)benzoate

The reactants of methyl 2-bromo-5-hydroxybenzoate (0.231 g, 1 mmol),3-methoxycarbonylphenyl boronic acid (0.214 g, 1.2 mmol), palladiumacetate (0.022 g, 0.1 mmol), potassium carbonate (0.346 g, 2.5 mmol) andtetrabutylammonium bromide (0.322 g, 1 mmol) in 1.2 ml of water washeated with stirring at 70° C. for 1 h and 20 mins in a sealed vial bymicrowave. Then the mixture was partitioned between AcOEt and 1 N HClaqueous solution. The organic layer was collected, dried with Na₂SO₄,then concentrated. The resulting residue was purified by silica gelchromatography with AcOEt/Hex combinations as eluent, giving the titlecompound (0.240 g) in 84% yield. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 10.02(s, 1H), 7.90 (td, J=2.03, 6.66 Hz, 1H), 7.72-7.81 (m, 1H), 7.45-7.60(m, 2H), 7.28 (d, J=8.52 Hz, 1H), 7.16 (d, J=2.47 Hz, 1H), 7.03 (dd,J=2.61, 8.38 Hz, 1H), 3.86 (s, 3H), 3.56 (s, 3H). MS (ESI): found[M+Na]+, 309.2.

Methyl2-(3-methoxycarbonylphenyl)-5-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-benzoate(6)

6 was prepared via glycosidation between α-D-mannose pentaacetate andmethyl 5-hydroxy-2-(3-methoxycarbonylphenyl)benzoate following theprocedure previously described.²⁵ Yield: 73%. ¹H NMR (300 MHz,METHANOL-d₄) δ ppm 3.56-3.68 (m, 4H) 3.68-3.84 (m, 3H) 3.86-3.98 (m, 4H)4.06 (dd, J=3.30, 1.92 Hz, 1H) 5.60 (d, J=1.92 Hz, 1H) 7.29-7.43 (m, 2H)7.45-7.53 (m, 2H) 7.56 (d, J=2.47 Hz, 1H) 7.84-7.92 (m, 1H) 7.93-8.04(m, 1H). MS (ESI): found [M+H]⁺, 449.0.

N1,N3-Dimethyl-5-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamide(7).N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-dicarboxamide(9)

dimethyl 5-bromobenzene-1,3-dicarboxylate (10.6 g, 36.8 mmol) wasdissolved in a 33 wt % solution of methylamine in EtOH (30 mL) andstirred for 6 h at RT. The precipitate that formed during the reactionwas filtered to give 5.3 g (53%) of the intermediate5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide as a white solid.Concentration of the remaining filtrate yielded an additional 4.6 g(46%) of product. 5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide (5.3g, 19.5 mmol), Pd(dppf)Cl₂ (0.87 g, 1.2 mmol), bis(pinacolato)diboron(6.1 g, 24 mmol), and potassium acetate (7.8 g, 80 mmol) were dissolvedin DMSO (100 mL). The solution was stirred under vacuum and thenrepressurized with nitrogen. This process was repeated 3 times and thenthe resultant mixture was stirred at 80° C. for 5 h under a nitrogenatmosphere. After removal of the solvent under high vacuum, the crudematerial was purified by silica gel chromatography to give 9 as a lighttan solid (2.2 g, 35%). ¹H NMR (300 MHz, DMSO-d6) □ ppm 8.63 (m, 2H),8.41 (t, J=1.51 Hz, 1H), 8.23 (d, J=1.65 Hz, 2H), 2.79 (d, J=4.40 Hz,6H), 1.33 (s, 12H). MS (ESI): found: [M+H]⁺, 319.2.

N1,N3-Dimethyl-5-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamide(7)

Using the procedure outlined in the synthesis of 5b with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-trifluoromethyl-phenoxy)tetrahydropyran-3-yl]acetate (0.57 g) andN1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-dicarboxamide(9) (0.48 g), the title compound was obtained (0.340 g) in 69% yield forthe two steps. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm 8.17-8.24 (m, 1H),8.14 (d, J=1.65 Hz, 2H), 7.92 (d, J=1.92 Hz, 1H), 7.87 (dd, J=2.20, 8.79Hz, 1H), 7.57 (d, J=8.79 Hz, 1H), 5.64 (d, J=1.65 Hz, 1H), 4.04 (dd,J=1.65, 3.30 Hz, 1H), 3.87-3.96 (dd, 1H), 3.64-3.83 (m, 3H), 3.48-3.63(m, 1H), 2.93 (s, 6H). MS (ESI): found: [M+H]⁺, 515.1.

N1,N3-Dimethyl-5-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamide(8)

8 was prepared using the same procedure as for 5b withN1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-dicarboxamideas the Suzuki coupling partner. Yield: 56%. ¹H NMR (300 MHz,METHANOL-d₄) δ □ppm 8.09-8.23 (m, 3H), 7.46-7.59 (m, 2H), 7.33 (d,J=8.52 Hz, 1H), 5.57 (d, J=1.92 Hz, 1H), 4.08 (dd, J=1.92, 3.30 Hz, 1H),3.97 (dd, J=3.43, 9.48 Hz, 1H), 3.67-3.85 (m, 3H), 3.60 (ddd, J=2.47,5.01, 7.35 Hz, 1H), 2.96 (s, 6H), 2.32 (s, 3H). MS (ESI): found: [M+H]⁺,461.2.

Procedure for the Preparation of Mannosides Via Amide Coupling ReactionN-(2-Hydroxyethyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzamide(10a)

Under nitrogen atmosphere, at 0° C. anhydrous DMF (5 mL) was added intothe RB flask containing 4′-(α-D-mannopyranosyloxy)biphenyl-3-carboxylicacid²⁵ (0.038 g, 0.1 mmol) and HATU (0.046 g, 0.12 mmol). After stirringfor 10 min, aminoethanol (0.007 g, 0.12 mmol), thenN,N-diisopropylethylamine (0.039 g, 0.3 mmol) were added. The mixturewas stirred overnight while being warmed to rt naturally. The solventwas removed and the residue was purified by HPLC (C18, 15*150 mm column;eluent: acetonitrile/water (0.1% TFA)) to give the title compound (0.032g) in 76% yield. ¹H NMR (300 MHz, DEUTERIUM OXIDE) δ ppm 3.53-3.63 (m,2H) 3.68-3.94 (m, 6H) 4.11 (dd, J=9.20, 3.43 Hz, 1H) 4.22 (dd, J=3.30,1.92 Hz, 1H) 5.66 (d, J=1.65 Hz, 1H) 7.21 (d, J=8.79 Hz, 2H) 7.47-7.62(m, 3H) 7.66-7.78 (m, 2H) 7.87 (t, J=1.65 Hz, 1H). MS (ESI): found[M+H]⁺, 420.1.

N-(2-Aminoethyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzamide(10b)

10b was prepared using the same procedure as for 10a. Yield: 60%. ¹H NMR(300 MHz, METHANOL-d₄) δ ppm 3.14-3.26 (m, 2H) 3.57-3.66 (m, 1H)3.66-3.83 (m, 5H) 3.87-4.00 (m, 1H) 4.03 (dd, J=3.30, 1.92 Hz, 1H)5.49-5.62 (m, 1H) 7.19-7.31 (m, 2H) 7.49-7.59 (m, 1H) 7.59-7.72 (m, 2H)7.75-7.89 (m, 2H) 8.03-8.21 (m, 1H). MS (ESI): found [M+H]⁺, 419.2.

Piperazin-1-yl-[3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]phenyl]methanone(10c)

10c was prepared using the same procedure as for 10a. Yield: 65%. ¹H NMR(300 MHz, METHANOL-d₄) δ ppm 3.54-3.67 (m, 1H) 3.67-3.85 (m, 4H)3.85-3.99 (m, 4H) 4.03 (dd, J=3.30, 1.92 Hz, 1H) 5.54 (d, J=1.65 Hz, 1H)7.19-7.29 (m, 2H) 7.42 (dt, J=7.69, 1.24 Hz, 1H) 7.50-7.64 (m, 3H)7.67-7.79 (m, 2H). MS (ESI): found [M+H]⁺, 445.3.

(4-Methylpiperazin-1-yl)-[3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]phenyl]methanone(10d)

10d was prepared using the same procedure as for 10a. Yield: 87%. ¹H NMR(300 MHz, METHANOL-d₄) δ ppm 2.96 (s, 3H) 3.053.65 (m, 9H) 3.68-3.85 (m,3H) 3.87-3.97 (m, 1H) 4.03 (dd, J=3.30, 1.92 Hz, 1H) 5.54 (d, J=1.65 Hz,1H) 7.17-7.30 (m, 2H) 7.42 (dt, J=7.62, 1.27 Hz, 1H) 7.50-7.66 (m, 3H)7.67-7.80 (m, 2H). MS (ESI): found [M+H]⁺, 459.0.

N-(4-Pyridyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzamide(10e)

10e was prepared using the same procedure as for 10a. Yield: 93%. ¹H NMR(300 MHz, METHANOL-d₄) δ ppm 3.57-3.69 (m, 1H) 3.69-3.84 (m, 3H) 3.93(dd, J=9.34, 3.30 Hz, 1H) 4.04 (dd, J=3.30, 1.92 Hz, 1H) 5.56 (d, J=1.65Hz, 1H) 7.18-7.37 (m, 2H) 7.57-7.78 (m, 3H) 7.82-8.06 (m, 2H) 8.24 (t,J=1.65 Hz, 1H) 8.36-8.51 (m, 2H) 8.68 (d, J=7.42 Hz, 2H). MS (ESI):found [M+H]⁺, 453.1.

N-(3-Pyridyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzamide(10f)

10f was prepared using the same procedure as for 10a. Yield: 75%. ¹H NMR(300 MHz, METHANOL-d₄) δ ppm 3.55-3.68 (m, 1H) 3.68-3.85 (m, 3H)3.88-3.98 (m, 1H) 4.04 (dd, J=3.43, 1.79 Hz, 1H) 5.56 (d, J=1.92 Hz, 1H)7.20-7.32 (m, 2H) 7.52-7.73 (m, 3H) 7.80-7.91 (m, 1H) 7.91-7.99 (m, 1H)8.04 (dd, J=8.65, 5.63 Hz, 1H) 8.23 (t, J=1.65 Hz, 1H) 8.59 (d, J=5.49Hz, 1H) 8.67-8.79 (m, 1H) 9.55 (s, 1H). MS (ESI): found [M+H]⁺, 453.2.

Procedure for the Preparation of Biphenyl Mannoside Derivatives ThroughSuzuki Coupling Reaction with[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]acetate (9) as Intermediates methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]pyridine-3-carboxylate(12b).[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]acetate (11)

Under nitrogen atmosphere, the mixture of 4-bromophenyl2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (2.791 g, 5.55 mmol),bis(pinacolato)diboron (1.690 g, 6.66 mmol), potassium acetate (2.177 g,22.18 mmol) and(1.1′-bis(diphenylphosohino)ferrocene)dichloropalladium(II) (0.244 g,0.33 mmol) in DMSO (50 ml) was heated at 80° C. with stirring for 2.5 h.The solvent was removed and the resulting residue was purified by silicagel chromography with hexane/ethyl acetate combinations as eluent togive 11 (2.48 g) in 81% yield. ¹H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.33(s, 12H) 1.98-2.12 (m, 9H) 2.20 (s, 3H) 3.93-4.19 (m, 2H) 4.21-4.36 (m,1H) 5.32-5.42 (m, 1H) 5.45 (dd, J=3.57, 1.92 Hz, 1H) 5.51-5.62 (m, 2H)7.00-7.15 (m, 2H) 7.67-7.84 (m, 2H). MS (ESI): found: [M+Na]⁺, 573.2.

Methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]pyridine-3-carboxylate(12b)

Under nitrogen atmosphere, the mixture of[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]acetate (0.132 g, 0.24 mmol), methyl 5-bromonicotinate (0.043 g, 0.2mmol), cesium carbonate (0.196 g, 0.6 mmol) andtetrakis(triphenylphosphine)palladium (0.023 g, 0.02 mmol) indioxane/water (5 mL/1 mL) was heated at 80° C. with stirring for 1 h.After cooling down, the mixture was filtered through silica gel columnto remove the metal catalyst and salts with hexane/ethyl acetate (2/1)containing 2% triethylamine as eluent. The filtrate was concentrated,then dried in vacuo. Into the residue, 6 mL of methanol with catalyticamount of sodium methoxide (0.02 M) was added and the mixture wasstirred at room temperature overnight. The solvent was removed. Theresulting residue was purified by silica gel chromography withCH₂Cl₂/MeOH combinations containing 2% NH₃/H₂O as eluent, giving rise to12b (0.031 g) in 40% yield. ¹H NMR (300 MHz, CD₃OD) b ppm 3.53-3.65 (m,1H) 3.67-3.83 (m, 3H) 3.89-3.96 (m, 1H) 3.99 (s, 3H) 4.04 (dd, J=3.43,1.79 Hz, 1H) 5.57 (d, J=1.92 Hz, 1H) 7.22-7.37 (m, 2H) 7.58-7.73 (m, 2H)8.54 (t, J=2.06 Hz, 1H) 8.97 (d, J=2.20 Hz, 1H) 9.04 (d, J=1.92 Hz, 1H).MS (ESI): found: [M+H]⁺, 392.1.

Methyl4-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]pyridine-2-carboxylate(12a)

12a was prepared using the same procedure as for 12b and was purified byHPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1% TFA)).Yield: 15%. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm 3.51-3.63 (m, 1H)3.65-3.84 (m, 3H) 3.88-3.95 (m, 1H) 4.00-4.13 (m, 4H) 5.62 (d, J=1.65Hz, 1H) 7.28-7.40 (m, 2H) 7.82-7.95 (m, 2H) 8.13 (dd, J=5.49, 1.92 Hz,1H) 8.55 (d, J=1.65 Hz, 1H) 8.73 (d, J=5.49 Hz, 1H). MS (ESI): found[M+H]⁺, 392.2.

Methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]-3-ureido-thiophene-2-carboxylate(13a)

13a was prepared using the same procedure as for 12b and was purified byHPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1% TFA)).Yield: 10%. ¹H NMR (300 MHz, METHANOL-d₄) δ ppm 3.58 (ddd, J=7.21, 4.88,2.47 Hz, 1H) 3.66-3.83 (m, 3H) 3.83-3.96 (m, 4H) 4.02 (dd, J=3.30, 1.92Hz, 1H) 5.54 (d, J=1.65 Hz, 1H) 7.12-7.27 (m, 2H) 7.56-7.69 (m, 2H) 8.12(s, 1H). MS (ESI): found [M+H]⁺, 455.1.

Methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]thiophene-2-carboxylate(13b)

13b was prepared using the same procedure as for 12b and was purified bysilica gel chromography with CH₂Cl₂/MeOH combinations. Yield: 23%. ¹HNMR (300 MHz, METHANOL-d₄) δ ppm 3.53-3.62 (m, 1H) 3.68-3.81 (m, 3H)3.85-3.94 (m, 4H) 4.02 (dd, J=3.60, 2.1 Hz, 1H) 5.54 (d, J=1.8 Hz, 1H)7.19 (m, 2H) 7.34 (d, J=3.9 Hz, 1H) 7.64 (m, 2H) 7.75 (d, J=3.9 Hz, 1H).MS (ESI): found [M+Na]+, 419.1.

(5-bromo-3-thienyl)urea (16)

Under nitrogen atmosphere N,N-diisopropylethylamine (0.390 g, 3 mmol)was added to the solution of 5-bromothiophene-3-carboxylic acid (0.207g, 1 mmol) and diphenylphosphoryl azide (DPPA) (0.330 g, 1.2 mmol) indioxane (5 ml) at rt. After stirring for 30 min, the mixture was heatedat 85 for 1.5 h. After the mixture cooled down to rt, 0.5 M of ammoniasolution in dioxane (12 ml) was added. 30 min's later, the solvents wasremoved and the resulting residue was purified by silica gelchromography with CH₂Cl₂/MeOH combinations to give(5-bromo-3-thienyl)urea (0.072 g) in 32% yield. ¹H NMR (300 MHz,DMSO-d₆) δ ppm 8.80 (s, 1H), 7.09 (s, 2H), 5.87 (s, 2H). MS (ESI): found[M+H]⁺, 223.0.

[5-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]-3-thienyl]urea(13c). 13c was prepared using the same procedure as for 12b and waspurified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water(0.1% TFA)). Yield: 80%. ¹H NMR (300 MHz, METHANOL-d₄/ACETONITRILE-d₃(3/1)) b 7.50-7.62 (m, 2H), 7.11-7.21 (m, 3H), 7.08 (d, J=1.37 Hz, 1H),5.53 (d, J=1.65 Hz, 1H), 4.02 (dd, J=1.92, 3.30 Hz, 1H), 3.82-3.95 (m,1H), 3.66-3.81 (m, 3H), 3.51-3.64 (m, 1H). MS (ESI): found [M+H]⁺,397.1.

Methyl5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]thiophene-3-carboxylate(13d)

13d was prepared using the same procedure as for 12b and was purified bysilica gel chromography with CH₂Cl₂/MeOH combinations. Yield: 33%. ¹HNMR (300 MHz, METHANOL-d₄) δ ppm 3.55-3.63 (m, 1H) 3.68-3.81 (m, 3H)3.84-3.94 (m, 4H) 4.02 (dd, J=3.30, 1.8 Hz, 1H) 5.53 (d, J=1.8 Hz, 1H)7.18 (m, 2H) 7.59 (m, 2H) 7.64 (d, J=1.5 Hz, 1H) 8.11 (d, J=1.5 Hz, 1H).MS (ESI): found [M+Na]⁺, 419.1.

(2R,3S,4S,5S,6R)-2-(Hydroxymethyl)-6-[4-(7-isoquinolyl)phenoxy]tetrahydropyran-3,4,5-triol(14a)

14a was prepared using the same procedure as for 12b and was purified byHPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1% TFA)).Yield: 73%. ¹H NMR (300 MHz, METHANOL-d₄) b 9.73 (s, 1H), 8.59-8.71 (m,1H), 8.47-8.58 (m, 2H), 8.42 (d, J=6.59 Hz, 1H), 8.33 (d, J=8.79 Hz,1H), 7.76-7.89 (m, 2H), 7.25-7.41 (m, 2H), 5.60 (d, J=1.92 Hz, 1H), 4.06(dd, J=1.92, 3.30 Hz, 1H), 3.87-4.00 (m, 1H), 3.68-3.87 (m, 3H),3.55-3.68 (m, 1H). MS (ESI): found [M+H]⁺, 384.2.

(2R,3S,4S,5S,6R)-2-(Hydroxymethyl)-6-(4-quinazolin-6-ylphenoxy)tetrahydropyran-3,4,5-triol(14b)

14b was prepared using the same procedure as for 12b. Yield: 28%. ¹H NMR(300 MHz, METHANOL-d₄) δ 9.51 (s, 1H), 9.15 (s, 1H), 8.21-8.35 (m, 2H),8.02 (d, J=8.52 Hz, 1H), 7.63-7.80 (m, J=8.79 Hz, 2H), 7.14-7.32 (m,J=8.79 Hz, 2H), 5.50 (d, J=1.37 Hz, 1H), 3.94-4.03 (m, 1H), 3.80-3.94(m, 1H), 3.62-3.80 (m, 3H), 3.48-3.62 (m, 1H). MS (ESI): found [M+H]⁺,385.1.

(2R,3S,4S,5S,6R)-2-(Hydroxymethyl)-6-[4-(5-isoquinolyl)phenoxy]tetrahydropyran-3,4,5-triol(15a)

15a was prepared using the same procedure as for 12b and was purified byHPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1% TFA)).Yield: 90%. ¹H NMR (300 MHz, METHANOL-d₄) b 9.80 (s, 1H), 8.45-8.60 (m,2H), 8.35 (d, J=6.59 Hz, 1H), 8.01-8.21 (m, 2H), 7.45-7.55 (m, 2H),7.31-7.42 (m, 2H), 5.62 (d, J=1.92 Hz, 1H), 4.07 (dd, J=1.92, 3.30 Hz,1H), 3.89-3.99 (m, 1H), 3.70-3.86 (m, 3H), 3.60-3.69 (m, 1H). MS (ESI):found [M+H]⁺, 384.2.

7-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]-2H-isoquinolin-1-one(15b)

15b was prepared using the same procedure as for 12b and was purified byHPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1% TFA)).Yield: 75%. ¹H NMR (300 MHz, METHANOL-d₄) b 8.51 (d, J=2.20 Hz, 1H),7.93-8.05 (m, 1H), 7.62-7.77 (m, 3H), 7.21-7.31 (m, 2H), 7.18 (d, J=7.14Hz, 1H), 6.71 (d, J=7.14 Hz, 1H), 5.56 (d, J=1.92 Hz, 1H), 4.04 (dd,J=1.92, 3.57 Hz, 1H), 3.88-4.00 (m, 1H), 3.69-3.87 (m, 3H), 3.57-3.69(m, 1H). MS (ESI): found [M+H]⁺, 400.2.

Affinity Measurement by Bio-Layer Interferometry.

Samples or buffer (200 μL per well) were dispensed into 96-wellmicrotiter plates (Greiner Bio-One, Monroe N.C.) and maintained at 30°C. with 1000RPM shaking. Pre-manufactured pins for individual assayswere made by biotinylating FimH lectin domain²⁵ at a 1:1 molar ratiowith NHS-PEO4-Biotin (Thermo Fisher, Rockford Ill.), diluting it to 50μg/ml in 20 mM HEPES pH 7.5, 150 mM NaCl (HBS), immobilizing it onhigh-binding streptavidin-coated biosensor tips (Super Streptavidin,FortéBio, Inc., Menlo Park, Calif.) for 10 minutes at 30° C., blockingthe pins with 10 μg/ml biocytin for 2 minutes, washing in HBS for 1hour, and rinsing in 15% sucrose in HBS. Pins were then air dried for 30minutes and stored in their original packaging with a dessicant packet.Assays were performed by re-wetting premade pins with HBS for 15minutes, then storing them in fresh HBS until use. Individual affinityassays were performed on an Octet Red instrument (Fort6Bio, Inc., MenloPark, Calif.) and consisted of a short baseline measurement followed byincubation of pins for 10 minutes with 7 twofold dilutions (in HBS) ofcompound in a concentration range experimentally determined to givewell-measured association and dissociation kinetics, then a 30-minutedissociation phase in HBS. Each experimental pin was referenced to abiocytin-blocked pin to control for instrument drift and a secondbiocytin-blocked pin that was passed through a duplicate experiment inthe same 96-well plate to control for nonspecific binding of thecompound to the pin. Kinetics data and affinity constants were generatedautomatically by the global fitting protocol in ForteBio Data Analysisversion 6.3. Typical signal for compound binding was approximately 0.2“nm shift” units, while the noise level of the instrument is around0.0025 nm.

Differential Scanning Fluorimetry (DSF) Method.

FimH lectin domain (residues 1-158 of UPEC J96 FimH, without an affinitytag) was purified as described previously. Five micrograms FimH in 5 μlHBS were mixed with HBS to yield a final volume of 50 μl containingcompound at a final concentration of 100 μM and a “5×” finalconcentration of SYPRO Orange (sold as a “5000×” stock in DMSO and mixedwith HBS to a working stock of 50× immediately before use: LifeTechnologies Inc.; Grand Island, N.Y.). Compounds were diluted to 100 μMfrom stocks in DMSO and compared to matched control wells with FimHalone plus 0.2% DMSO. 50 μl reactions were placed in 96-wellclear-bottom PCR plates and subjected to a melt curve from 20-90° C. in0.5° C. increments of 15 seconds each followed by a fluorescence read ofthe “HEX” channel in a Bio-Rad CFX96 thermocycler (Bio-Rad, Hercules,Calif.). Melt curves were fitted to the Boltzmann equation to determinethe melting temperature (T_(m)) (y=A2+(A1−A2)/(1+exp((x−x0)/dx)) wherex0 is the T_(m)) using OriginPro 8 (OriginLab, Northampton Mass.).

Pharmacokinetic Studies.

Compound levels in mouse urine and plasma were made using an AB SciexAPI-4000 QTrap (AB Sciex, Foster City, Calif.) as previouslydescribed.³⁶ Selected reaction monitoring (SRM) mode quantification wasperformed with using the following MS/MS transitions [precursormass/charge ratio (m/z)/product m/z]: compound 3, 447/285; compound 5a,424/262; compound 5b, 404/242; compound 5c, 548/296; compound 7,515/353; compound 8, 461/299; compound 3 R group, 285/254.

Parallel Artificial Membrane Permeability Assay (PAMPA).

Materials.

The assay was carried out using Multiscreen PVDF 96-well plates(Millipore, Billerica, Mass.) using the company's Transporter ReceiverPlate. The lipid (dioleyoylphosphotidylcholine (DOPC):Stearic Acid(80:20, wt %) in dodecane was obtained from Avanti Polar lipids(Alabaster, Ala.). Hank's Buffered Salt Solution (HBSS), pH 7.4 wasobtained from MediaTech (Manassas, Va.).

Methods.

Each of the test compounds was diluted to 2.5 mM in DMSO (Sigma, StLouis, Mo.) and further diluted prior to testing to 2.5 μM in HBSS, pH7.4. The assay was performed using a Millipore 96-well Multiscreen-IPPAMPA plate, 5 μl of the lipid suspension was directly added to the PVDFmembrane of the filter plate. Immediately following the addition of thelipid to the membrane, 200 μl of HBSS solution containing the testcompound was added to the donor (upper) chamber. HBSS (300 PI) is alsoadded to the receiver plate and the filter and receiver plates assembledand incubated overnight at room temperature in a moistened sealed bag toprevent evaporation. The concentration in the receiver plate as well asan equilibrium plate, that represents a theoretical, partition-freesample, was determined by HPLC-tandem mass spectrometry. Analysis ofeach compound was performed in triplicate.

Analysis.

Analysis of both the acceptor and equilibrium samples were performedusing an AB 3200 triple quadripole mass spectrometer linked to aShimadzu DGU-20A HPLC with a Prevail C18 column (3 μm, 2.1×10 mm) with aflow rate of 0.35 ml/min. The mobile phase used was A: 0.1% Formic Acidin water, B: 0.1% Formic Acid in methanol. The elation gradient methodis described in the table. Data acquisition and peak heightdetermination was performed using Analyst v.1.4.2.

Time, min A B 0.01 95 5 0.50 95 5 1.00 5 95 2.00 5 95 2.01 95 5 6.01Stop

Calculations.

The log₁₀ of the effective permeability (Log P_(e)) was calculated usingthe following equation

${\log\; P_{e}} = {{\log\left\{ {{C \cdot {- \ln}}\mspace{11mu}\left( {1 - \frac{\lbrack{drug}\rbrack_{acceptor}}{\lbrack{drug}\rbrack_{equilibrium}}} \right)} \right\}\mspace{14mu}{where}\mspace{14mu} C} = \left( \frac{V_{D} \cdot V_{A}}{\left( {V_{D} + {V_{A}\mspace{14mu}{{Area} \cdot {Time}}}} \right)} \right)}$Where the drug concentration is the peak areas for the analyte and V_(D)and V_(A) is the volume of the donor and acceptor compartmentrespectively. The area is the surface area of the PVDF membrane (0.11cm²) and time is the incubation in seconds (64,800 sec). Each value isthe mean of triplicates performed on the same day.Introduction for Examples 37-39

Type 1 pili constitute the major UPEC virulence factor, being criticalfor attachment to and invasion of the bladder epithelium as well as forIBC formation. Type 1 pili belong to a class of extracellular fibersassembled by the chaperone-usher pathway. They are encoded by the fimgene cluster, and their expression is directed by a phase-variablepromoter (fimS), which facilitates a switch between piliated andnonpiliated bacterial states. UPEC populations are heterogeneousconsisting of bacteria that are bald, low-, moderately- and highlypiliated. The ratio of each piliated fraction shifts depending on theenvironment; studies investigating the expression of type 1 pilirevealed that UPEC associated with epithelial cells are highly piliated,consistent with the critical role of type 1 pili in urotheliumcolonization, while bacteria recovered from urine samples of patientshave fimS primarily in the OFF phase and are likely non-piliated.Regulation of fimS phase-variation is controlled by the FimB, FimE andFimX recombinases, the expression of which is in turn influenced bynumerous regulatory factors. We recently identified the QseC sensorkinase as one of the factors implicated in regulation of type 1 piliexpression. In the qseC mutant, the fim promoter is found primarily inthe OFF orientation, resulting in reduced type 1 pili expression, aneffect that stems from uncontrolled activation of the QseB responseregulator in this mutant. In addition to influencing type 1 piliexpression in the absence of qseC, over-active QseB aberrantly regulatesconserved cellular pathways and impacts several virulence-associatedgenes, resulting in UPEC attenuation.

Numerous investigations highlighted the importance of type 1 pili inpathogenesis and spurred the onset of research towards the developmentof anti-adhesion agents, known as mannosides, aimed at inhibitingFimH-host receptor interactions. Given that disruption of QseCsimultaneously affects fim expression and a number of other cellularprocesses, type 1 pili were disengaged from QseC control to determinethe infection stages at which other QseC-mediated defects becomeimportant. By locking the invertible fim promoter element in the ONorientation, type 1 pili expression was restored in the qseC mutant andit was shown that the resulting mutant is competent in infectioninitiation and IBC formation. However, even upon expression of type 1pili the qseC mutant is rapidly outcompeted by the parent strain duringco-infection studies and cannot survive long-term in the urinary tractin mono-infections. These findings indicate that although type 1 pilimask the qseC deletion phenotypes at the onset of infection, additionalQseC-mediated processes are required for persistence. Therefore, theexamples below explore whether co-inhibition of QseC and type 1 piliwould have a synergistic effect on clearing UPEC from the urinary tract.Given the lack of available QseC inhibitors a prophylactic approach wastaken, using the qseC mutant as a proxy to QseC-inhibition. The datashowed that mice pre-treated with mannoside (to block FimH-mediatedadhesion) and subsequently challenged with the qseC mutant were moreeffectively protected against chronic UTI compared to mice challengedwith the parent strain. These examples indicate that targeting of QseCcan be exploited as a potential preventative strategy, alone or incombination with other anti-virulence agents.

Example 37. Deletion of QseC Impairs the Ability of UPEC to Invade theBladder

It was previously shown that deletion of qseC in UTI89 leads to reducedbladder bacterial titers during the acute stages of infection (6 and 16h post infection) in a cystitis murine model. Given that type 1 pili arecritical for bladder adherence and invasion and are significantlyreduced in the absence of QseC, it was rationalized that the in vivoattenuation of UTI89ΔqseC could be, at least partly, attributed toreduced bacterial internalization. Thus, the ability of UTI89ΔqseC toadhere to and invade the bladder epithelium was assessed and compared tothe parent strain. Female C3H/HeN mice were transurethrally inoculatedwith 10′ wild-type (wt) UTI89 or UTI89ΔqseC and bacterial invasion wasassessed at 1 and 3 h post infection (h.p.i), time-points previouslyshown to be sufficient for completion of the invasion events.Enumeration of colony forming units (cfu) recovered from infectedbladders indicated that compared to wt UTI89, UTI89ΔqseC hadsignificantly lower overall bacterial titers (intracellular and luminal)at both time-points (FIG. 52A). Treatment of infected bladders withgentamicin to eradicate the luminal bacterial population but leave theintracellular population unharmed revealed decreased intracellularnumbers for UTI89ΔqseC (FIG. 52B). Thus, these data indicate thatdeletion of qseC affects the ability of UTI89 to initiate infection bycompromising its ability to colonize and invade the host bladder.

Example 38. Expression of Type 1 Pili Rescues the Adherence Propertiesof UTI89ΔqseC In Vitro, but does not Influence Other ΔqseC-RelatedDefects

Attenuation of UTI89ΔqseC is linked to reduced bacterial internalizationdue to decreased type 1 pili production. Therefore, it was investigatedwhether restoration of type 1 pili expression alone would be sufficientto overcome the in vivo defects of UTI89ΔqseC. It was previouslyreported that in the absence of QseC the phase-variable fim promoterdriving type 1 pili expression, fimS, is primarily switched to the OFForientation. Therefore, fimS was locked in the ON orientation in thechromosome of wt UTI89 and UTI89ΔqseC to attain expression of type 1pili independently of QseC (FIG. 53A). Electron microscopy revealed thatthe resulting strains, UTI89_LON and UTI89ΔqseC_LON, were nothyper-piliated, but rather a larger population of piliated versusnon-piliated bacteria was observed compared to wt UTI89 (data notshown). UTI89_LON and UTI89ΔqseC_LON exhibited comparablemannose-sensitive hemagglutination (HA) properties, which were higherthan wt UTI89 since inversion of the promoter to the OFF state can nolonger occur (FIG. 53B). Consistently, western blot analyses probing forFimA, the major type 1 pilus subunit, verified that type 1 piliproduction in UTI89ΔqseC_LON is comparable to UTI89_LON, but again,higher than wt UTI89 (FIG. 53C).

Example 39. Dual Inhibition of Type 1 Pili and QseC Potentiates UPECElimination from the Bladder

Type 1 pili are sufficient for establishment of acute infection whereasQseC is a critical determinant for persistence in the bladder; thustargeting both factors could constitute an excellent means ofpotentiating UPEC clearance from the urinary tract by interfering withboth the acute and chronic aspects of infection. Mannosides, compoundsthat bind with high affinity the mannose-binding pocket of the FimHadhesin at the tip of type 1 pili, are potent inhibitors of type 1 pilifunction. In particular, mannosides 6 and 8, the most potent mannosidestested, drastically reduce bacterial titers within 6 hours of oraldelivery when used prophylactically or as a therapeutic strategy. Thesestudies are a significant step towards a new anti-bacterial therapeutic;however, a fraction of the bacterial population persists after mannosidetreatment and could lead to relapse, suggesting that mannosideeffectiveness can be further optimized. It was therefore rationalizedthat co-inhibition of QseC may enhance mannoside efficacy. However, noQseC inhibitors are currently available. Although previous work hasidentified LED209 as an inhibitor of the kinase activity of QseC, It hasbeen established that it is the disruption of the QseC phosphataseproperty that is responsible for the attenuation of a qseC mutant. Tocircumvent the lack of available QseC-specific phosphatase inhibitors,UTI89ΔqseC was used as a proxy to QseC inhibition and a prophylacticstrategy was employed to investigate whether targeting both factors(type 1 pili and QseC) could effectively protect the host against UTI.Mice were pretreated with mannoside 6, and infected 30 min posttreatment with wt UTI89 or UTI89ΔqseC. Mice treated with PBS prior toinfection were included as controls. Bladder bacterial titers wereenumerated at 2 weeks post infection as a measure of chronic infection.Compared to non-treated mice, there was a 2-log reduction inmannoside-treated mice infected with wt UTI89 (FIG. 54, UTI89 vs.UTI89_MAN) that was similar to the reduction observed in non-treatedmice infected with UTI89ΔqseC (FIG. 54, UTI89_MAN vs. UTI89ΔqseC).Notably, in mannoside-treated animals infected with UTI89ΔqseC,bacterial reduction was further enhanced, as indicated by an additional1.5-log drop compared to mannoside-treated animals infected with wtUTI89 (FIG. 54, UTI89_MAN vs. UTI89ΔqseC_MAN). These data stronglyindicate that dual targeting of type 1 pili and QseC could potentiateUPEC elimination from the host bladder.

Materials and Methods for Examples 37-39

Strains, Constructs and Growth Conditions

UTI89_LON and UTI89ΔqseC_LON were created using, Red Recombinase, so asto mutate 7 out of 9 nucleotides in the left invertible repeat of thefim promoter in the chromosome. Bacteria were incubated in Luria Bertani(LB) media at 37° C. for 4 h under shaking conditions, sub-cultured(1:1000) in fresh LB media and incubated statically at 37° C. for 18 h.

Mouse Infections

Female C3H/HeN mice (Harlan), 7-9 weeks old, were used for all studiesdescribed below and in each case mice were infected with 107 bacteria.

Short-Term Infections:

Mice were transurethrally infected with bacteria carrying theGFP-expressing plasmid pCom-GFP, as previously described, and sacrificedat 1 and 3 h.p.i. Bladders were aseptically removed, homogenized andplated for total bacterial enumeration or gentamicin-treated todetermine intracellular bacterial titers. Experiment was repeated 3times.

Ex Vivo Cqentamicin Assay:

Bladders were bisected and washed 3 times in 500 μl PBS. The washes werecollected and plated for cfu enumeration, to determine luminal bacteria.Washed bladders were incubated for 90 min at 37° C. with 100 μg/mlgentamicin to kill adherent extracellular bacteria. Following wash (3×)with PBS, bladders were homogenized in 1 ml PBS and plated to determineintracellular bacterial titers. Two-tailed Mann-Whitney (P<0.05) wasused for statistical analyses.

Acute Infection Studies:

Mice were transurethrally infected with 107 bacteria carrying theplasmid pCom-GFP as previously described. Experiments were repeatedthree times and statistically analyzed using two-tailed Mann-Whitney(P<0.05, considered significant).

Long-Term Infection Studies:

Mice were infected with chromosomally marked strains of wt UTI89,UTI89_LON, UTI89ΔqseC or UTI89ΔqseC_LON. Mice were sacrificed at 2 weekspost inoculation and organs were processed for cfu enumeration on LB andLB/Kanamycin agar plates. Experiment was repeated 2 times. Cumulativedata from all experiments are presented.

Immunoblots, HA and Phase Assays

Bacteria were grown statically in LB for 18 h at 37° C. Immunoblots(using anti-type 1 pili antibody), HA and phase assays were performed onnormalized cells (OD₆₀₀=1) as previously described.

qPCR Analyses

RNA extraction, DNase treatment and reverse transcription were preformedusing reagents and methods as reported by Kostakioti et al. Relativetranscript abundance was determined by qPCR as previously describedusing aceB- or qseB-primers.

The invention claimed is:
 1. A compound of structural Formula XXIV

wherein: X is selected from the group consisting of hydrogen, OD², SD², and ND^(z); Z is O⁴; Y is oxygen⁴; D², D⁴, D⁵ are independently selected from the group consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl; n is an integer from 1 to 10; A is independently selected from the group consisting of C, CD⁶ and N; D⁶ is independently selected from the group consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl; D²⁴ is selected from the group consisting of hydrogen, halogen, hydrocarbyl, and substituted hydrocarbyl; D²⁵ and D²⁶ is selected from the group consisting of hydrogen, —NHCONH₂, —COOMe, and —CONHMe, or D²⁵ and D²⁶ can optionally form a cycloalkyl or heterocyclo ring; D^(z) is independently selected from the group consisting of hydrogen hydrocarbyl, substituted hydrocarbyl, —COD^(x), —COND^(x)D^(x)SO₂D^(x), and —CO₂D^(x); and D^(x) is independently selected from the group consisting of hydrogen, —ND⁴D⁵, or an optionally substituted alkyl, cycloalkyl, heterocycle, or aryl.
 2. The compound of claim 1, wherein X is OR²; and Z is O.
 3. The compound of claim 1, wherein D²⁴ is hydrocarbyl.
 4. The compound of claim 3, wherein D²⁴ is methyl.
 5. The compound of claim 1, wherein D²⁵ and D²⁶ form a cycloalkyl or heterocyclo ring.
 6. A method of treating a urinary tract infection, the method comprising administering a compound of claim 1 to a subject in need thereof.
 7. The method of claim 6, wherein the subject is further administered a bactericidal composition.
 8. A method of reducing the resistance of a bacterium to a bactericidal compound, the method comprising administering a compound of claim 1 to a subject in need thereof. 